Antibody constructs for egfrviii and cd3

ABSTRACT

The present invention relates to a bispecific antibody construct comprising a first binding domain which binds to human EGFRVIII on the surface of a target cell and a second binding domain which binds to human CD3 on the surface of a T cell. Moreover, the invention provides a polynucleotide encoding the antibody construct, a vector comprising said polynucleotide and a host cell transformed or transfected with said polynucleotide or vector. Furthermore, the invention provides a process for the production of the antibody construct of the invention, a medical use of said antibody construct and a kit comprising said antibody construct.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/225,627, filed Aug. 1, 2016, now issued U.S. Pat. No.10,519,241, which claims priority to U.S. Provisional Patent ApplicationNo. 62/290,861, filed on Feb. 3, 2016 and U.S. Provisional PatentApplication No. 62/199,945 filed Jul. 31, 2015, which are eachincorporated by reference herein in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of the disclosure, aSequence Listing in computer readable form (filename:49899B_SeqListing.txt; 349,275 bytes; created Nov. 6, 2019), which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a bispecific antibody constructcomprising a first binding domain which binds to human EGFRVIII on thesurface of a target cell and a second binding domain which binds tohuman CD3 on the surface of a T cell. Moreover, the invention provides apolynucleotide encoding the antibody construct, a vector comprising saidpolynucleotide and a host cell transformed or transfected with saidpolynucleotide or vector. Furthermore, the invention provides a processfor the production of the antibody construct of the invention, a medicaluse of said antibody construct and a kit comprising said antibodyconstruct.

BACKGROUND

EGFRvIII is one of several EGFR variants that are caused by generearrangement accompanied by EGFR gene amplification. EGFRvIII consistsof a 267 amino acid in-frame deletion in the extracellular domain ofEGFR. EGFRvIII is the most commonly occurring variant of the epidermalgrowth factor (EGF) receptor in human cancers. During the process ofgene amplification, a 267 amino acid deletion occurs in theextracellular domain creating a novel junction which can serve as tumorspecific neo epitope for monoclonal antibodies. This variant of the EGFreceptor contributes to tumor progression through constitutive signalingin a ligand independent manner. EGFRvIII is not known to be expressed onany normal tissues. The excellent tumor selectivity of EGFRvIIIexpression establishes EGFRvIII as an ideal antigen for targeting with aBiTE antibody construct. Contribution of EGFRvIII to tumor progressionsuggests a dependence of cancer cells on expression of EGFRvIII andtherefore further supports its suitability as a target.

It has been shown that EGFRvIII is frequently expressed in two types ofmalignant central nervous system tumors, namely Glioblastoma Multiformeand Anaplastic Astrocytoma. For both diseases there exists a significantunmet medical need. This is exemplified by the poor overall survivalunder standard of care with 13.6% at 2 years for Glioblastoma Multiformeand 25.9% at 5 years for Anaplastic Astrocytoma. Currently the standardof care for Glioblastoma Multiforme consists of surgical resection ofthe tumor, which is most often hindered by the diffuse growth pattern ofthe tumor and the requirement to preserve functionally essential regionsof the brain. Surgery is followed by adjuvant irradiation andchemotherapy. For Glioblastoma Multiforme and Anaplastic Astrocytomatreated according to the current standard of care recurrence is thenorm, with eventually fatal outcome in virtually all patients.

EGFRvIII expression in Glioblastoma Multiforme and AnaplasticAstrocytoma constitutively activates the PI3 kinase signaling pathwayand is associated with worsened prognosis in Glioblastoma Multiforme andAnaplastic Astrocytoma. Also it has been described that EGFRvIII iscoexpressed with CD133 and defines a population of cancer stem cells inGlioblastoma Multiforme (Emlet et al., Cancer Res, 2014, 74(4):1238-49).Furthermore it has been demonstrated that by the mechanism of anintercellular antigen transfer EGFRvIII can be transferred to the cellsurface of antigen negative tumor cells. By this mechanism expressionheterogeneity of EGFRvIII, which has been shown for some cases ofGlioblastoma Multiforme, might be overcome as an obstacle for treatmentefficacy, especially in the case of using an EGFRvIII specific BiTEantibody construct, which provides a highly effective cytotoxic mode ofaction compensating for potentially low levels of target antigenachieved by intercellular antigen transfer.

EGFRvIII expression has also been described in several other tumorentities (Wikstrand, C J. et al., Cancer Research 55(14): 3140-3148(1995); Ge H. et al., Int J Cancer. 98(3):357-61 (2002); Moscatello, G.et al., Cancer Res. 55(23):5536-9 (1995); Garcia de Palazzo, I E. etal., Cancer Res. 53(14):3217-20 (1993); Olapade-Olaopa, E O. et al., BrJ Cancer. 82(1): 186-94 (2000)). Therefore, given the exquisite tumorselectivity of EGFRvIII, treatment with an EGFRvIII specific BiTEantibody construct might also be beneficial in other, select cancertypes or subtypes. Potentially selection of cancer types, subtypes orpatients for treatment could be guided by various methods testing forexpression of EGFRvIII in the tumor.

As there is still a need for having available further options for thetreatment of solid tumor diseases related to the overexpression ofEGFRVIII, such as glioblastoma, astrocytoma, medulloblastomas, breastcarcinomas, non-small cell lung carcinomas, ovarian carcinomas, prostatecarcinomas, central nervous system, there are provided herewith meansand methods for the solution of this problem in the form of a bispecificantibody construct having a binding domain directed to EGFRVIII on thesurface of tumor target cells and a second binding domain directed toCD3 on the surface of T cells.

SUMMARY AND DETAILED DESCRIPTION

Thus, in a first aspect, the present invention provides a bispecificantibody construct comprising a first binding domain which binds tohuman and macaque EGFRVIII on the surface of a target cell and a secondbinding domain which binds to human CD3 on the surface of a T cell,wherein the first binding domain comprises a polypeptide as depicted inSEQ ID NO: 157 and a polypeptide as depicted in SEQ ID NO: 158.

It must be noted that as used herein, the singular forms “a”, “an”, and“the” include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within ±20%,preferably within ±15%, more preferably within ±10%, and most preferablywithin ±5% of a given value or range.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

The term “antibody construct” refers to a molecule in which thestructure and/or function is/are based on the structure and/or functionof an antibody, e.g., of a full-length or whole immunoglobulin molecule.An antibody construct is hence capable of binding to its specific targetor antigen. Furthermore, an antibody construct according to theinvention comprises the minimum structural requirements of an antibodywhich allow for the target binding. This minimum requirement may e.g. bedefined by the presence of at least the three light chain CDRs (i.e.CDR1, CDR2 and CDR3 of the VL region) and/or the three heavy chain CDRs(i.e. CDR1, CDR2 and CDR3 of the VH region), preferably of all six CDRs.The antibodies on which the constructs according to the invention arebased include for example monoclonal, recombinant, chimeric,deimmunized, humanized and human antibodies.

Within the definition of “antibody constructs” according to theinvention are full-length or whole antibodies also including camelidantibodies and other immunoglobulin antibodies generated bybiotechnological or protein engineering methods or processes. Thesefull-length antibodies may be for example monoclonal, recombinant,chimeric, deimmunized, humanized and human antibodies. Also within thedefinition of “antibody constructs” are fragments of full-lengthantibodies, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab′, F(ab′)2 or“r IgG” (“half antibody”). Antibody constructs according to theinvention may also be modified fragments of antibodies, also calledantibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc,scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies,tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv,“minibodies” exemplified by a structure which is as follows:(VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or(scFv-CH3-scFv)2, multibodies such as triabodies or tetrabodies, andsingle domain antibodies such as nanobodies or single variable domainantibodies comprising merely one variable domain, which might be VHH, VHor VL, that specifically bind an antigen or epitope independently ofother V regions or domains.

A binding domain may typically comprise an antibody light chain variableregion (VL) and an antibody heavy chain variable region (VH); however,it does not have to comprise both. Fd fragments, for example, have twoVH regions and often retain some antigen-binding function of the intactantigen-binding domain. Additional examples for the format of antibodyfragments, antibody variants or binding domains include (1) a Fabfragment, a monovalent fragment having the VL, VH, CL and CH1 domains;(2) a F(ab′)2 fragment, a bivalent fragment having two Fab fragmentslinked by a disulfide bridge at the hinge region; (3) an Fd fragmenthaving the two VH and CH1 domains; (4) an Fv fragment having the VL andVH domains of a single arm of an antibody, (5) a dAb fragment (Ward etal., (1989) Nature 341:544-546), which has a VH domain; (6) an isolatedcomplementarity determining region (CDR), and (7) a single chain Fv(scFv), the latter being preferred (for example, derived from anscFV-library). Examples for embodiments of antibody constructs accordingto the invention are e.g. described in WO 00/006605, WO 2005/040220, WO2008/119567, WO 2010/037838, WO 2013/026837, WO 2013/026833, US2014/0308285, US 2014/0302037, W 02014/144722, WO 2014/151910, and WO2015/048272.

Furthermore, the definition of the term “antibody construct” includesmonovalent, bivalent and polyvalent/multivalent constructs and, thus,monospecific constructs, specifically binding to only one antigenicstructure, as well as bispecific and polyspecific/multispecificconstructs, which specifically bind more than one antigenic structure,e.g. two, three or more, through distinct binding domains. Moreover, thedefinition of the term “antibody construct” includes moleculesconsisting of only one polypeptide chain as well as molecules consistingof more than one polypeptide chain, which chains can be either identical(homodimers, homotrimers or homo oligomers) or different (heterodimer,heterotrimer or heterooligomer). Examples for the above identifiedantibodies and variants or derivatives thereof are described inter aliain Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988)and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermannand Dubel, Antibody Engineering, Springer, 2nd ed. 2010 and Little,Recombinant Antibodies for Immunotherapy, Cambridge University Press2009.

The antibody constructs of the present invention are preferably “invitro generated antibody constructs”. This term refers to an antibodyconstruct according to the above definition where all or part of thevariable region (e.g., at least one CDR) is generated in a non-immunecell selection, e.g., an in vitro phage display, protein chip or anyother method in which candidate sequences can be tested for theirability to bind to an antigen. This term thus preferably excludessequences generated solely by genomic rearrangement in an immune cell inan animal. A “recombinant antibody” is an antibody made through the useof recombinant DNA technology or genetic engineering.

The term “monoclonal antibody” (mAb) or monoclonal antibody construct asused herein refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations and/or post-translation modifications (e.g.,isomerizations, amidations) that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site or determinant on the antigen, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (orepitopes). In addition to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,hence uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod.

For the preparation of monoclonal antibodies, any technique providingantibodies produced by continuous cell line cultures can be used. Forexample, monoclonal antibodies to be used may be made by the hybridomamethod first described by Koehler et al., Nature, 256: 495 (1975), ormay be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). Examples for further techniques to produce human monoclonalantibodies include the trioma technique, the human B-cell hybridomatechnique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985), 77-96).

Hybridomas can then be screened using standard methods, such asenzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance(BIACORE™) analysis, to identify one or more hybridomas that produce anantibody that specifically binds with a specified antigen. Any form ofthe relevant antigen may be used as the immunogen, e.g., recombinantantigen, naturally occurring forms, any variants or fragments thereof,as well as an antigenic peptide thereof. Surface plasmon resonance asemployed in the BIAcore system can be used to increase the efficiency ofphage antibodies which bind to an epitope of a target antigen, such asEGFRVIII or CD3 epsilon (Schier, Human Antibodies Hybridomas 7 (1996),97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

Another exemplary method of making monoclonal antibodies includesscreening protein expression libraries, e.g., phage display or ribosomedisplay libraries. Phage display is described, for example, in Ladner etal., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317,Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol.Biol., 222: 581-597 (1991).

In addition to the use of display libraries, the relevant antigen can beused to immunize a non-human animal, e.g., a rodent (such as a mouse,hamster, rabbit or rat). In one embodiment, the non-human animalincludes at least a part of a human immunoglobulin gene. For example, itis possible to engineer mouse strains deficient in mouse antibodyproduction with large fragments of the human Ig (immunoglobulin) loci.Using the hybridoma technology, antigen-specific monoclonal antibodiesderived from the genes with the desired specificity may be produced andselected. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics7:13-21, US 2003-0070185, WO 96/34096, and WO 96/33735.

A monoclonal antibody can also be obtained from a non-human animal, andthen modified, e.g., humanized, deimmunized, rendered chimeric etc.,using recombinant DNA techniques known in the art. Examples of modifiedantibody constructs include humanized variants of non-human antibodies,“affinity matured” antibodies (see, e.g. Hawkins et al. J. Mol. Biol.254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837(1991)) and antibody mutants with altered effector function(s) (see,e.g., U.S. Pat. No. 5,648,260, Kontermann and Dubel (2010), loc. cit.and Little (2009), loc. cit.).

In immunology, affinity maturation is the process by which B cellsproduce antibodies with increased affinity for antigen during the courseof an immune response. With repeated exposures to the same antigen, ahost will produce antibodies of successively greater affinities. Likethe natural prototype, the in vitro affinity maturation is based on theprinciples of mutation and selection. The in vitro affinity maturationhas successfully been used to optimize antibodies, antibody constructs,and antibody fragments. Random mutations inside the CDRs are introducedusing radiation, chemical mutagens or error-prone PCR. In addition, thegenetical diversity can be increased by chain shuffling. Two or threerounds of mutation and selection using display methods like phagedisplay usually results in antibody fragments with affinities in the lownanomolar range.

A preferred type of an amino acid substitutional varianation of theantibody constructs involves substituting one or more hypervariableregion residues of a parent antibody (e. g. a humanized or humanantibody). Generally, the resulting variant(s) selected for furtherdevelopment will have improved biological properties relative to theparent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e. g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibody variants thus generated are displayed in amonovalent fashion from filamentous phage particles as fusions to thegene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e. g. binding affinity) as herein disclosed. In order to identifycandidate hypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the bindingdomain and, e.g., human EGFRVIII. Such contact residues and neighbouringresidues are candidates for substitution according to the techniqueselaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening as described herein and antibodieswith superior properties in one or more relevant assays may be selectedfor further development.

The monoclonal antibodies and antibody constructs of the presentinvention specifically include “chimeric” antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc.Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primitized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc.) and human constant region sequences. Avariety of approaches for making chimeric antibodies have beendescribed. See e.g., Morrison et al., Proc. Natl. Acad. ScL U.S.A.81:6851 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S.Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi etal., EP 0171496; EP 0173494; and GB 2177096.

An antibody, antibody construct, antibody fragment or antibody variantmay also be modified by specific deletion of human T cell epitopes (amethod called “deimmunization”) by the methods disclosed for example inWO 98/52976 or WO 00/34317. Briefly, the heavy and light chain variabledomains of an antibody can be analyzed for peptides that bind to MHCclass II; these peptides represent potential T cell epitopes (as definedin WO 98/52976 and WO 00/34317). For detection of potential T cellepitopes, a computer modeling approach termed “peptide threading” can beapplied, and in addition a database of human MHC class II bindingpeptides can be searched for motifs present in the VH and VL sequences,as described in WO 98/52976 and WO 00/34317. These motifs bind to any ofthe 18 major MHC class II DR allotypes, and thus constitute potential Tcell epitopes. Potential T cell epitopes detected can be eliminated bysubstituting small numbers of amino acid residues in the variabledomains, or preferably, by single amino acid substitutions. Typically,conservative substitutions are made. Often, but not exclusively, anamino acid common to a position in human germline antibody sequences maybe used. Human germline sequences are disclosed e.g. in Tomlinson, etal. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995) Immunol.Today Vol. 16 (5): 237-242; and Tomlinson et al. (1995) EMBO J. 14:14:4628-4638. The V BASE directory provides a comprehensive directory ofhuman immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al. MRC Centre for Protein Engineering, Cambridge, UK). Thesesequences can be used as a source of human sequence, e.g., for frameworkregions and CDRs. Consensus human framework regions can also be used,for example as described in U.S. Pat. No. 6,300,064.

“Humanized” antibodies, antibody constructs, variants or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-bindingsubsequences of antibodies) are antibodies or immunoglobulins of mostlyhuman sequences, which contain (a) minimal sequence(s) derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region (also CDR) of the recipient are replaced byresidues from a hypervariable region of a non-human (e.g., rodent)species (donor antibody) such as mouse, rat, hamster or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, “humanized antibodies”as used herein may also comprise residues which are found neither in therecipient antibody nor the donor antibody. These modifications are madeto further refine and optimize antibody performance. The humanizedantibody may also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321: 522-525 (1986);Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op.Struct. Biol., 2: 593-596 (1992).

Humanized antibodies or fragments thereof can be generated by replacingsequences of the Fv variable domain that are not directly involved inantigen binding with equivalent sequences from human Fv variabledomains. Exemplary methods for generating humanized antibodies orfragments thereof are provided by Morrison (1985) Science 229:1202-1207;by Oi et al. (1986) BioTechniques 4:214; and by U.S. Pat. Nos.5,585,089; 5,693,761; 5,693,762; 5,859,205; and 6,407,213. Those methodsinclude isolating, manipulating, and expressing the nucleic acidsequences that encode all or part of immunoglobulin Fv variable domainsfrom at least one of a heavy or light chain. Such nucleic acids may beobtained from a hybridoma producing an antibody against a predeterminedtarget, as described above, as well as from other sources. Therecombinant DNA encoding the humanized antibody molecule can then becloned into an appropriate expression vector.

Humanized antibodies may also be produced using transgenic animals suchas mice that express human heavy and light chain genes, but areincapable of expressing the endogenous mouse immunoglobulin heavy andlight chain genes. Winter describes an exemplary CDR grafting methodthat may be used to prepare the humanized antibodies described herein(U.S. Pat. No. 5,225,539). All of the CDRs of a particular humanantibody may be replaced with at least a portion of a non-human CDR, oronly some of the CDRs may be replaced with non-human CDRs. It is onlynecessary to replace the number of CDRs required for binding of thehumanized antibody to a predetermined antigen.

A humanized antibody can be optimized by the introduction ofconservative substitutions, consensus sequence substitutions, germlinesubstitutions and/or back mutations. Such altered immunoglobulinmolecules can be made by any of several techniques known in the art,(e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983;Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth.Enzymol., 92: 3-16, 1982, and EP 239 400).

The term “human antibody”, “human antibody construct” and “human bindingdomain” includes antibodies, antibody constructs and binding domainshaving antibody regions such as variable and constant regions or domainswhich correspond substantially to human germline immunoglobulinsequences known in the art, including, for example, those described byKabat et al. (1991) (Ioc. cit.). The human antibodies, antibodyconstructs or binding domains of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs, and inparticular, in CDR3. The human antibodies, antibody constructs orbinding domains can have at least one, two, three, four, five, or morepositions replaced with an amino acid residue that is not encoded by thehuman germline immunoglobulin sequence. The definition of humanantibodies, antibody constructs and binding domains as used herein alsocontemplates fully human antibodies, which include only non-artificiallyand/or genetically altered human sequences of antibodies as those can bederived by using technologies or systems such as the Xenomouse.

In some embodiments, the antibody constructs of the invention are“isolated” or “substantially pure” antibody constructs. “Isolated” or“substantially pure”, when used to describe the antibody constructsdisclosed herein, means an antibody construct that has been identified,separated and/or recovered from a component of its productionenvironment. Preferably, the antibody construct is free or substantiallyfree of association with all other components from its productionenvironment. Contaminant components of its production environment, suchas that resulting from recombinant transfected cells, are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. The antibody constructs may e.g constituteat least about 5%, or at least about 50% by weight of the total proteinin a given sample. It is understood that the isolated protein mayconstitute from 5% to 99.9% by weight of the total protein content,depending on the circumstances. The polypeptide may be made at asignificantly higher concentration through the use of an induciblepromoter or high expression promoter, such that it is made at increasedconcentration levels. The definition includes the production of anantibody construct in a wide variety of organisms and/or host cells thatare known in the art. In preferred embodiments, the antibody constructwill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Ordinarily, however, an isolated antibody construct willbe prepared by at least one purification step.

The term “binding domain” characterizes in connection with the presentinvention a domain which (specifically) binds to/interactswith/recognizes a given target epitope or a given target site on thetarget molecules (antigens), here: EGFRVIII and CD3, respectively. Thestructure and function of the first binding domain (recognizingEGFRVIII), and preferably also the structure and/or function of thesecond binding domain (recognizing CD3), is/are based on the structureand/or function of an antibody, e.g. of a full-length or wholeimmunoglobulin molecule. According to the invention, the first bindingdomain is characterized by the presence of three light chain CDRs (i.e.CDR1, CDR2 and CDR3 of the VL region) and/or three heavy chain CDRs(i.e. CDR1, CDR2 and CDR3 of the VH region). The second binding domainpreferably also comprises the minimum structural requirements of anantibody which allow for the target binding. More preferably, the secondbinding domain comprises at least three light chain CDRs (i.e. CDR1,CDR2 and CDR3 of the VL region) and/or three heavy chain CDRs (i.e.CDR1, CDR2 and CDR3 of the VH region). It is envisaged that the firstand/or second binding domain is produced by or obtainable byphage-display or library screening methods rather than by grafting CDRsequences from a pre-existing (monoclonal) antibody into a scaffold.

According to the present invention, binding domains are in the form ofone or more polypeptides. Such polypeptides may include proteinaceousparts and non-proteinaceous parts (e.g. chemical linkers or chemicalcross-linking agents such as glutaraldehyde). Proteins (includingfragments thereof, preferably biologically active fragments, andpeptides, usually having less than 30 amino acids) comprise two or moreamino acids coupled to each other via a covalent peptide bond (resultingin a chain of amino acids). The term “polypeptide” as used hereindescribes a group of molecules, which usually consist of more than 30amino acids. Polypeptides may further form multimers such as dimers,trimers and higher oligomers, i.e., consisting of more than onepolypeptide molecule. Polypeptide molecules forming such dimers, trimersetc. may be identical or non-identical. The corresponding higher orderstructures of such multimers are, consequently, termed homo- orheterodimers, homo- or heterotrimers etc. An example for ahereteromultimer is an antibody molecule, which, in its naturallyoccurring form, consists of two identical light polypeptide chains andtwo identical heavy polypeptide chains. The terms “peptide”,“polypeptide” and “protein” also refer to naturally modifiedpeptides/polypeptides/proteins wherein the modification is effected e.g.by post-translational modifications like glycosylation, acetylation,phosphorylation and the like. A “peptide”, “polypeptide” or “protein”when referred to herein may also be chemically modified such aspegylated. Such modifications are well known in the art and describedherein below.

Preferably the binding domain which binds to EGFRVIII and/or the bindingdomain which binds to CD3 is/are human binding domains. Antibodies andantibody constructs comprising at least one human binding domain avoidsome of the problems associated with antibodies or antibody constructsthat possess non-human such as rodent (e.g. murine, rat, hamster orrabbit) variable and/or constant regions. The presence of such rodentderived proteins can lead to the rapid clearance of the antibodies orantibody constructs or can lead to the generation of an immune responseagainst the antibody or antibody construct by a patient. In order toavoid the use of rodent derived antibodies or antibody constructs, humanor fully human antibodies/antibody constructs can be generated throughthe introduction of human antibody function into a rodent so that therodent produces fully human antibodies.

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the use of such technology for substitution ofmouse loci with their human equivalents could provide unique insightsinto the expression and regulation of human gene products duringdevelopment, their communication with other systems, and theirinvolvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B-cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (mAbs)—an important milestone towards fulfilling the promiseof antibody therapy in human disease. Fully human antibodies or antibodyconstructs are expected to minimize the immunogenic and allergicresponses intrinsic to mouse or mouse-derivatized mAbs and thus toincrease the efficacy and safety of the administered antibodies/antibodyconstructs. The use of fully human antibodies or antibody constructs canbe expected to provide a substantial advantage in the treatment ofchronic and recurring human diseases, such as inflammation,autoimmunity, and cancer, which require repeated compoundadministrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human mAbs with thedesired specificity could be readily produced and selected. This generalstrategy was demonstrated in connection with the generation of the firstXenoMouse mouse strains (see Green et al. Nature Genetics 7:13-21(1994)). The XenoMouse strains were engineered with yeast artificialchromosomes (YACs) containing 245 kb and 190 kb-sized germlineconfiguration fragments of the human heavy chain locus and kappa lightchain locus, respectively, which contained core variable and constantregion sequences. The human Ig containing YACs proved to be compatiblewith the mouse system for both rearrangement and expression ofantibodies and were capable of substituting for the inactivated mouse Iggenes. This was demonstrated by their ability to induce B celldevelopment, to produce an adult-like human repertoire of fully humanantibodies, and to generate antigen-specific human mAbs. These resultsalso suggested that introduction of larger portions of the human Ig locicontaining greater numbers of V genes, additional regulatory elements,and human Ig constant regions might recapitulate substantially the fullrepertoire that is characteristic of the human humoral response toinfection and immunization. The work of Green et al. was recentlyextended to the introduction of greater than approximately 80% of thehuman antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci andkappa light chain loci, respectively. See Mendez et al. Nature Genetics15:146-156 (1997) and U.S. patent application Ser. No. 08/759,620.

The production of the XenoMouse mice is further discussed and delineatedin U.S. patent application Ser. No. 07/466,008, Ser. No. 07/610,515,Ser. No. 07/919,297, Ser. No. 07/922,649, Ser. No. 08/031,801, Ser. No.08/112,848, Ser. No. 08/234,145, Ser. No. 08/376,279, Ser. No.08/430,938, Ser. No. 08/464,584, Ser. No. 08/464,582, Ser. No.08/463,191, Ser. No. 08/462,837, Ser. No. 08/486,853, Ser. No.08/486,857, Ser. No. 08/486,859, Ser. No. 08/462,513, Ser. No.08/724,752, and Ser. No. 08/759,620; and U.S. Pat. Nos. 6,162,963;6,150,584; 6,114,598; 6,075,181, and 5,939,598 and Japanese Patent Nos.3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also Mendez et al.Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med.188:483-495 (1998), EP 0 463 151 B1, WO 94/02602, WO 96/34096, WO98/24893, WO 00/76310, and WO 03/47336.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more VH genes, one ormore DH genes, one or more JH genes, a mu constant region, and a secondconstant region (preferably a gamma constant region) are formed into aconstruct for insertion into an animal. This approach is described inU.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806;5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650;5,814,318; 5,877,397; 5,874,299; and 6,255,458 each to Lonberg and Kay,U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfort and Berns, U.S.Pat. Nos. 5,612,205; 5,721,367; and U.S. Pat. No. 5,789,215 to Berns etal., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, Ser. No.07/575,962, Ser. No. 07/810,279, Ser. No. 07/853,408, Ser. No.07/904,068, Ser. No. 07/990,860, Ser. No. 08/053,131, Ser. No.08/096,762, Ser. No. 08/155,301, Ser. No. 08/161,739, Ser. No.08/165,699, Ser. No. 08/209,741. See also EP 0 546 073 B1, WO 92/03918,WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No.5,981,175. See further Taylor et al. (1992), Chen et al. (1993),Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994),Taylor et al. (1994), and Tuaillon et al. (1995), Fishwild et al.(1996).

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961. Xenerex Biosciences is developinga technology for the potential generation of human antibodies. In thistechnology, SCID mice are reconstituted with human lymphatic cells,e.g., B and/or T cells. Mice are then immunized with an antigen and cangenerate an immune response against the antigen. See U.S. Pat. Nos.5,476,996; 5,698,767; and 5,958,765.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. It is howeverexpected that certain human anti-chimeric antibody (HACA) responses willbe observed, particularly in chronic or multi-dose utilizations of theantibody. Thus, it would be desirable to provide antibody constructscomprising a human binding domain against EGFRVIII and a human bindingdomain against CD3 in order to vitiate concerns and/or effects of HAMAor HACA response.

The terms “(specifically) binds to”, (specifically) recognizes”, “is(specifically) directed to”, and “(specifically) reacts with” mean inaccordance with this invention that a binding domain interacts orspecifically interacts with a given epitope or a given target site onthe target molecules (antigens), here: EGFRVIII and CD3, respectively.

The term “epitope” refers to a site on an antigen to which a bindingdomain, such as an antibody or immunoglobulin, or a derivative, fragmentor variant of an antibody or an immunoglobulin, specifically binds. An“epitope” is antigenic and thus the term epitope is sometimes alsoreferred to herein as “antigenic structure” or “antigenic determinant”.Thus, the binding domain is an “antigen interaction site”. Saidbinding/interaction is also understood to define a “specificrecognition”.

“Epitopes” can be formed both by contiguous amino acids ornon-contiguous amino acids juxtaposed by tertiary folding of a protein.A “linear epitope” is an epitope where an amino acid primary sequencecomprises the recognized epitope. A linear epitope typically includes atleast 3 or at least 4, and more usually, at least 5 or at least 6 or atleast 7, for example, about 8 to about 10 amino acids in a uniquesequence.

A “conformational epitope”, in contrast to a linear epitope, is anepitope wherein the primary sequence of the amino acids comprising theepitope is not the sole defining component of the epitope recognized(e.g., an epitope wherein the primary sequence of amino acids is notnecessarily recognized by the binding domain). Typically aconformational epitope comprises an increased number of amino acidsrelative to a linear epitope. With regard to recognition ofconformational epitopes, the binding domain recognizes athree-dimensional structure of the antigen, preferably a peptide orprotein or fragment thereof (in the context of the present invention,the antigenic structure for one of the binding domains is comprisedwithin the EGFRVIII protein). For example, when a protein molecule foldsto form a three-dimensional structure, certain amino acids and/or thepolypeptide backbone forming the conformational epitope becomejuxtaposed enabling the antibody to recognize the epitope. Methods ofdetermining the conformation of epitopes include, but are not limitedto, x-ray crystallography, two-dimensional nuclear magnetic resonance(2D-NMR) spectroscopy and site-directed spin labelling and electronparamagnetic resonance (EPR) spectroscopy.

A method for epitope mapping is described in the following: When aregion (a contiguous amino acid stretch) in the human EGFRVIII proteinis exchanged/replaced with its corresponding region of a non-human andnon-primate EGFRVIII antigen (e.g., mouse EGFRVIII, but others likechicken, rat, hamster, rabbit etc. might also be conceivable), adecrease in the binding of the binding domain is expected to occur,unless the binding domain is cross-reactive for the non-human,non-primate EGFRVIII used. Said decrease is preferably at least 10%,20%, 30%, 40%, or 50%; more preferably at least 60%, 70%, or 80%, andmost preferably 90%, 95% or even 100% in comparison to the binding tothe respective region in the human EGFRVIII protein, whereby binding tothe respective region in the human EGFRVIII protein is set to be 100%.

A further method to determine the contribution of a specific residue ofa target antigen to the recognition by a antibody construct or bindingdomain is alanine scanning (see e.g. Morrison K L & Weiss G A. Cur OpinChem Biol. 2001 June; 5(3):302-7), where each residue to be analyzed isreplaced by alanine, e.g. via site-directed mutagenesis. Alanine is usedbecause of its non-bulky, chemically inert, methyl functional group thatnevertheless mimics the secondary structure references that many of theother amino acids possess. Sometimes bulky amino acids such as valine orleucine can be used in cases where conservation of the size of mutatedresidues is desired. Alanine scanning is a mature technology which hasbeen used for a long period of time.

The interaction between the binding domain and the epitope or the regioncomprising the epitope implies that a binding domain exhibitsappreciable affinity for the epitope/the region comprising the epitopeon a particular protein or antigen (here: EGFRVIII and CD3,respectively) and, generally, does not exhibit significant reactivitywith proteins or antigens other than EGFRVIII or CD3. “Appreciableaffinity” includes binding with an affinity of about 10⁻⁶ M (KD) orstronger. Preferably, binding is considered specific when the bindingaffinity is about 10⁻¹² to 10⁻⁸ M, 10⁻¹² to 10⁻⁹ M, 10⁻¹² to 10⁻¹⁰ M,10⁻¹¹ to 10⁻⁸ M, preferably of about 10⁻¹¹ to 10⁻⁹ M. Whether a bindingdomain specifically reacts with or binds to a target can be testedreadily by, inter alia, comparing the reaction of said binding domainwith a target protein or antigen with the reaction of said bindingdomain with proteins or antigens other than EGFRVIII or CD3. Preferably,a binding domain of the invention does not essentially or substantiallybind to proteins or antigens other than EGFRVIII or CD3 (i.e., the firstbinding domain is not capable of binding to proteins other than EGFRVIIIand the second binding domain is not capable of binding to proteinsother than CD3).

The term “does not essentially/substantially bind” or “is not capable ofbinding” means that a binding domain of the present invention does notbind a protein or antigen other than EGFRVIII or CD3, i.e., does notshow reactivity of more than 30%, preferably not more than 20%, morepreferably not more than 10%, particularly preferably not more than 9%,8%, 7%, 6% or 5% with proteins or antigens other than EGFRVIII or CD3,whereby binding to EGFRVIII or CD3, respectively, is set to be 100%.

Specific binding is believed to be effected by specific motifs in theamino acid sequence of the binding domain and the antigen. Thus, bindingis achieved as a result of their primary, secondary and/or tertiarystructure as well as the result of secondary modifications of saidstructures. The specific interaction of the antigen-interaction-sitewith its specific antigen may result in a simple binding of said site tothe antigen. Moreover, the specific interaction of theantigen-interaction-site with its specific antigen may alternatively oradditionally result in the initiation of a signal, e.g. due to theinduction of a change of the conformation of the antigen, anoligomerization of the antigen, etc.

The epitope of the bispeciric antibody construct of the invention islocated at the EGFRvIII specific junction between amino acid residues 5and 274 of EGFR, which results from the mutation of EGFR toward thesplice mutation EGFRvIII. This mutation removes residues 2-273 of themature EGFR sequence while it introduces a single glycine residue (seeFIG. 1)

It is also preferred in one embodiment of the invention that the secondbinding domain binds to human CD3 epsilon and to Callithrix jacchus,Saguinus Oedipus or Saimiri sciureus CD3 epsilon.

The term “variable” refers to the portions of the antibody orimmunoglobulin domains that exhibit variability in their sequence andthat are involved in determining the specificity and binding affinity ofa particular antibody (i.e., the “variable domain(s)”). The pairing of avariable heavy chain (VH) and a variable light chain (VL) together formsa single antigen-binding site.

Variability is not evenly distributed throughout the variable domains ofantibodies; it is concentrated in sub-domains of each of the heavy andlight chain variable regions. These sub-domains are called“hypervariable regions” or “complementarity determining regions” (CDRs).The more conserved (i.e., non-hypervariable) portions of the variabledomains are called the “framework” regions (FRM or FR) and provide ascaffold for the six CDRs in three dimensional space to form anantigen-binding surface. The variable domains of naturally occurringheavy and light chains each comprise four FRM regions (FR1, FR2, FR3,and FR4), largely adopting a β-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the β-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRM and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site (see Kabat et al., loc. cit.).

The terms “CDR”, and its plural “CDRs”, refer to the complementaritydetermining region of which three make up the binding character of alight chain variable region (CDR-L1, CDR-L2 and CDR-L3) and three makeup the binding character of a heavy chain variable region (CDR-H1,CDR-H2 and CDR-H3). CDRs contain most of the residues responsible forspecific interactions of the antibody with the antigen and hencecontribute to the functional activity of an antibody molecule: they arethe main determinants of antigen specificity.

The exact definitional CDR boundaries and lengths are subject todifferent classification and numbering systems. CDRs may therefore bereferred to by Kabat, Chothia, contact or any other boundarydefinitions, including the numbering system described herein. Despitediffering boundaries, each of these systems has some degree of overlapin what constitutes the so called “hypervariable regions” within thevariable sequences. CDR definitions according to these systems maytherefore differ in length and boundary areas with respect to theadjacent framework region. See for example Kabat (an approach based oncross-species sequence variability), Chothia (an approach based oncrystallographic studies of antigen-antibody complexes), and/orMacCallum (Kabat et al., loc. cit.; Chothia et al., J. Mol. Biol, 1987,196: 901-917; and MacCallum et al., J. Mol. Biol, 1996, 262: 732). Stillanother standard for characterizing the antigen binding site is the AbMdefinition used by Oxford Molecular's AbM antibody modeling software.See, e.g., Protein Sequence and Structure Analysis of Antibody VariableDomains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg). To the extent that tworesidue identification techniques define regions of overlapping, but notidentical regions, they can be combined to define a hybrid CDR. However,the numbering in accordance with the so-called Kabat system ispreferred.

Typically, CDRs form a loop structure that can be classified as acanonical structure. The term “canonical structure” refers to the mainchain conformation that is adopted by the antigen binding (CDR) loops.From comparative structural studies, it has been found that five of thesix antigen binding loops have only a limited repertoire of availableconformations. Each canonical structure can be characterized by thetorsion angles of the polypeptide backbone. Correspondent loops betweenantibodies may, therefore, have very similar three dimensionalstructures, despite high amino acid sequence variability in most partsof the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothiaet al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996,263: 800). Furthermore, there is a relationship between the adopted loopstructure and the amino acid sequences surrounding it. The conformationof a particular canonical class is determined by the length of the loopand the amino acid residues residing at key positions within the loop,as well as within the conserved framework (i.e., outside of the loop).Assignment to a particular canonical class can therefore be made basedon the presence of these key amino acid residues.

The term “canonical structure” may also include considerations as to thelinear sequence of the antibody, for example, as catalogued by Kabat(Kabat et al., loc. cit.). The Kabat numbering scheme (system) is awidely adopted standard for numbering the amino acid residues of anantibody variable domain in a consistent manner and is the preferredscheme applied in the present invention as also mentioned elsewhereherein. Additional structural considerations can also be used todetermine the canonical structure of an antibody. For example, thosedifferences not fully reflected by Kabat numbering can be described bythe numbering system of Chothia et al. and/or revealed by othertechniques, for example, crystallography and two- or three-dimensionalcomputational modeling. Accordingly, a given antibody sequence may beplaced into a canonical class which allows for, among other things,identifying appropriate chassis sequences (e.g., based on a desire toinclude a variety of canonical structures in a library). Kabat numberingof antibody amino acid sequences and structural considerations asdescribed by Chothia et al., loc. cit. and their implications forconstruing canonical aspects of antibody structure, are described in theliterature. The subunit structures and three-dimensional configurationsof different classes of immunoglobulins are well known in the art. For areview of the antibody structure, see Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

The CDR3 of the light chain and, particularly, the CDR3 of the heavychain may constitute the most important determinants in antigen bindingwithin the light and heavy chain variable regions. In some antibodyconstructs, the heavy chain CDR3 appears to constitute the major area ofcontact between the antigen and the antibody. In vitro selection schemesin which CDR3 alone is varied can be used to vary the binding propertiesof an antibody or determine which residues contribute to the binding ofan antigen. Hence, CDR3 is typically the greatest source of moleculardiversity within the antibody-binding site. H3, for example, can be asshort as two amino acid residues or greater than 26 amino acids.

In a classical full-length antibody or immunoglobulin, each light (L)chain is linked to a heavy (H) chain by one covalent disulfide bond,while the two H chains are linked to each other by one or more disulfidebonds depending on the H chain isotype. The CH domain most proximal toVH is usually designated as CH1. The constant (“C”) domains are notdirectly involved in antigen binding, but exhibit various effectorfunctions, such as antibody-dependent, cell-mediated cytotoxicity andcomplement activation. The Fc region of an antibody is comprised withinthe heavy chain constant domains and is for example able to interactwith cell surface located Fc receptors.

The sequence of antibody genes after assembly and somatic mutation ishighly varied, and these varied genes are estimated to encode 10¹⁰different antibody molecules (Immunoglobulin Genes, 2^(nd) ed., eds.Jonio et al., Academic Press, San Diego, Calif., 1995). Accordingly, theimmune system provides a repertoire of immunoglobulins. The term“repertoire” refers to at least one nucleotide sequence derived whollyor partially from at least one sequence encoding at least oneimmunoglobulin. The sequence(s) may be generated by rearrangement invivo of the V, D, and J segments of heavy chains, and the V and Jsegments of light chains. Alternatively, the sequence(s) can begenerated from a cell in response to which rearrangement occurs, e.g.,in vitro stimulation. Alternatively, part or all of the sequence(s) maybe obtained by DNA splicing, nucleotide synthesis, mutagenesis, andother methods, see, e.g., U.S. Pat. No. 5,565,332. A repertoire mayinclude only one sequence or may include a plurality of sequences,including ones in a genetically diverse collection.

The term “bispecific” as used herein refers to an antibody constructwhich is “at least bispecific”, ie., it comprises at least a firstbinding domain and a second binding domain, wherein the first bindingdomain binds to one antigen or target (here: EGFRVIII), and the secondbinding domain binds to another antigen or target (here: CD3).Accordingly, antibody constructs according to the invention comprisespecificities for at least two different antigens or targets. The term“bispecific antibody construct” of the invention also encompassesmultispecific antibody constructs such as trispecific antibodyconstructs, the latter ones including three binding domains, orconstructs having more than three (e.g. four, five . . . ) specificites.

Given that the antibody constructs according to the invention are (atleast) bispecific, they do not occur naturally and they are markedlydifferent from naturally occurring products. A “bispecific” antibodyconstruct or immunoglobulin is hence an artificial hybrid antibody orimmunoglobulin having at least two distinct binding sites with differentspecificities. Bispecific antibody constructs can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.79:315-321 (1990).

The at least two binding domains and the variable domains of theantibody construct of the present invention may or may not comprisepeptide linkers (spacer peptides). The term “peptide linker” comprisesin accordance with the present invention an amino acid sequence by whichthe amino acid sequences of one (variable and/or binding) domain andanother (variable and/or binding) domain of the antibody construct ofthe invention are linked with each other. An essential technical featureof such peptide linker is that it does not comprise any polymerizationactivity. Among the suitable peptide linkers are those described in U.S.Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344. The peptide linkerscan also be used to attach other domains or modules or regions (such ashalf-life extending domains) to the antibody construct of the invention.

In the event that a linker is used, this linker is preferably of alength and sequence sufficient to ensure that each of the first andsecond domains can, independently from one another, retain theirdifferential binding specificities. For peptide linkers which connectthe at least two binding domains (or two variable domains) in theantibody construct of the invention, those peptide linkers are preferredwhich comprise only a few number of amino acid residues, e.g. 12 aminoacid residues or less. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6or 5 amino acid residues are preferred. An envisaged peptide linker withless than 5 amino acids comprises 4, 3, 2 or one amino acid(s), whereinGly-rich linkers are preferred. A particularly preferred “single” aminoacid in the context of said “peptide linker” is Gly. Accordingly, saidpeptide linker may consist of the single amino acid Gly. Anotherpreferred embodiment of a peptide linker is characterized by the aminoacid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly₄Ser (SEQ ID NO: 1), orpolymers thereof, i.e. (Gly₄Ser)x, where x is an integer of 1 or greater(e.g. 2 or 3). Preferred linkers are depicted in SEQ ID NOs: 1-9. Thecharacteristics of said peptide linker, which comprise the absence ofthe promotion of secondary structures, are known in the art and aredescribed e.g. in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273),Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow(FASEB (1995) 9(1), 73-80). Peptide linkers which furthermore do notpromote any secondary structures are preferred. The linkage of saiddomains to each other can be provided, e.g., by genetic engineering, asdescribed in the examples. Methods for preparing fused and operativelylinked bispecific single chain constructs and expressing them inmammalian cells or bacteria are well-known in the art (e.g. WO 99/54440or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).

As described herein above, the invention provides a preferred embodimentwherein the antibody construct is in a format selected from the groupconsisting of (scFv)₂, scFv-single domain mAb, diabodies and oligomersof any of the those formats.

According to a particularly preferred embodiment, and as documented inthe appended examples, the antibody construct of the invention is a“bispecific single chain antibody construct”, more prefereably abispecific “single chain Fv” (scFv). Although the two domains of the Fvfragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker—as describedhereinbefore—that enables them to be made as a single protein chain inwhich the VL and VH regions pair to form a monovalent molecule; seee.g., Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883).These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are evaluatedfor function in the same manner as are whole or full-length antibodies.A single-chain variable fragment (scFv) is hence a fusion protein of thevariable region of the heavy chain (VH) and of the light chain (VL) ofimmunoglobulins, usually connected with a short linker peptide of aboutten to about 25 amino acids, preferably about 15 to 20 amino acids. Thelinker is usually rich in glycine for flexibility, as well as serine orthreonine for solubility, and can either connect the N-terminus of theVH with the C-terminus of the VL, or vice versa. This protein retainsthe specificity of the original immunoglobulin, despite removal of theconstant regions and introduction of the linker.

Bispecific single chain molecules are known in the art and are describedin WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS,(1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45,193-197, Loffler, Blood, (2000), 95, 6, 2098-2103, Bruhl, Immunol.,(2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56.Techniques described for the production of single chain antibodies (see,inter alia, U.S. Pat. No. 4,946,778, Kontermann and Dbel (2010), loc.cit. and Little (2009), loc. cit.) can be adapted to produce singlechain antibody constructs specifically recognizing (an) electedtarget(s).

Bivalent (also called divalent) or bispecific single-chain variablefragments (bi-scFvs or di-scFvs having the format (scFv)₂ can beengineered by linking two scFv molecules (e.g. with linkers as describedhereinbefore). If these two scFv molecules have the same bindingspecificity, the resulting (scFv)₂ molecule will preferably be calledbivalent (i.e. it has two valences for the same target epitope). If thetwo scFv molecules have different binding specificities, the resulting(scFv)₂ molecule will preferably be called bispecific. The linking canbe done by producing a single peptide chain with two VH regions and twoVL regions, yielding tandem scFvs (see e.g. Kufer P. et al., (2004)Trends in Biotechnology 22(5):238-244). Another possibility is thecreation of scFv molecules with linker peptides that are too short forthe two variable regions to fold together (e.g. about five amino acids),forcing the scFvs to dimerize. This type is known as diabodies (see e.g.Hollinger, Philipp et al., (July 1993) Proceedings of the NationalAcademy of Sciences of the United States of America 90 (14): 6444-8.).

Accorcing to an also preferred embodiment of the antibody construct ofthe invention the heavy chain (VH) and of the light chain (VL) of abinding domain binding either to the targe antigen EGFRVIII or CD3 arenot directly connected via an above described peptide linker but thebinding domain is formed due to the formation of a bispecifc molecule asdescribed for the diabody. Thus, the VH chain of the CD3 binding domainmay be fused to the VL of the EGFRVIII binding domain via such peptidelinker, while the VH chain of the EGFRVIII binding domain is fused tothe VL of the CD3 binding domain via such peptide linker.

Single domain antibodies comprise merely one (monomeric) antibodyvariable domain which is able to bind selectively to a specific antigen,independently of other V regions or domains. The first single domainantibodies were engineered from havy chain antibodies found in camelids,and these are called V_(H)H fragments. Cartilaginous fishes also haveheavy chain antibodies (IgNAR) from which single domain antibodiescalled V_(NAR) fragments can be obtained. An alternative approach is tosplit the dimeric variable domains from common immunoglobulins e.g. fromhumans or rodents into monomers, hence obtaining VH or VL as a singledomain Ab. Although most research into single domain antibodies iscurrently based on heavy chain variable domains, nanobodies derived fromlight chains have also been shown to bind specifically to targetepitopes. Examples of single domain antibodies are called sdAb,nanobodies or single variable domain antibodies.

A (single domain mAb)₂ is hence a monoclonal antibody construct composedof (at least) two single domain monoclonal antibodies, which areindividually selected from the group comprising VH, VL, V_(H)H andV_(NAR). The linker is preferably in the form of a peptide linker.Similarly, an “scFv-single domain mAb” is a monoclonal antibodyconstruct composed of at least one single domain antibody as describedabove and one scFv molecule as described above. Again, the linker ispreferably in the form of a peptide linker.

T cells or T lymphocytes are a type of lymphocyte (itself a type ofwhite blood cell) that play a central role in cell-mediated immunity.There are several subsets of T cells, each with a distinct function. Tcells can be distinguished from other lymphocytes, such as B cells andNK cells, by the presence of a T cell receptor (TCR) on the cellsurface. The TCR is responsible for recognizing antigens bound to majorhistocompatibility complex (MHC) molecules and is composed of twodifferent protein chains. In 95% of the T cells, the TCR consists of analpha (a) and beta (β) chain. When the TCR engages with antigenicpeptide and MHC (peptide/MHC complex), the T lymphocyte is activatedthrough a series of biochemical events mediated by associated enzymes,co-receptors, specialized adaptor molecules, and activated or releasedtranscription factors

The CD3 receptor complex is a protein complex and is composed of fourchains. In mammals, the complex contains a CD3γ (gamma) chain, a CD3δ(delta) chain, and two CD3ε (epsilon) chains. These chains associatewith the T cell receptor (TCR) and the so-called ζ (zeta) chain to formthe T cell receptor CD3 complex and to generate an activation signal inT lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chainsare highly related cell-surface proteins of the immunoglobulinsuperfamily containing a single extracellular immunoglobulin domain. Theintracellular tails of the CD3 molecules contain a single conservedmotif known as an immunoreceptor tyrosine-based activation motif or ITAMfor short, which is essential for the signaling capacity of the TCR. TheCD3 epsilon molecule is a polypeptide which in humans is encoded by theCD3ε gene which resides on chromosome 11. The most preferred epitope ofCD3 epsilon is comprised within amino acid residues 1-27 of the humanCD3 epsilon extracellular domain.

The redirected lysis of target cells via the recruitment of T cells by amultispecific, at least bispecific, antibody construct involvescytolytic synapse formation and delivery of perforin and granzymes. Theengaged T cells are capable of serial target cell lysis, and are notaffected by immune escape mechanisms interfering with peptide antigenprocessing and presentation, or clonal T cell differentiation; see, forexample, WO 2007/042261.

Cytotoxicity mediated by EGFRVII×CD3 bispecific antibody constructs canbe measured in various ways. See Example 1. Effector cells can be e.g.stimulated enriched (human) CD8 positive T cells or unstimulated (human)peripheral blood mononuclear cells (PBMC). If the target cells are ofmacaque origin or express or are transfected with macaque EGFRVIII, theeffector cells should also be of macaque origin such as a macaque T cellline, e.g. 4119LnPx. The target cells should express (at least theextracellular domain of) EGFRVIII, e.g. human or macaque EGFRVIII.Target cells can be a cell line (such as CHO) which is stably ortransiently transfected with EGFRVIII, e.g. human or macaque EGFRVIII.Alternatively, the target cells can be a EGFRVIII positive naturalexpresser cell line, such as the human glioblastoma cell line U87 ORDK-MG. Usually EC50 values are expected to be lower with target celllines expressing higher levels of EGFRVIII on the cell surface. Theeffector to target cell (E:T) ratio is usually about 10:1, but can alsovary. Cytotoxic activity of EGFRVII×CD3 bispecific antibody constructscan be measured in a 51-chromium release assay (incubation time of about18 hours) or in a in a FACS-based cytotoxicity assay (incubation time ofabout 48 hours). Modifications of the assay incubation time (cytotoxicreaction) are also possible. Other methods of measuring cytotoxicity arewell-known to the skilled person and comprise MTT or MTS assays,ATP-based assays including bioluminescent assays, the sulforhodamine B(SRB) assay, WST assay, clonogenic assay and the ECIS technology.

The cytotoxic activity mediated by EGFRVII×CD3 bispecific antibodyconstructs of the present invention is preferably measured in acell-based cytotoxicity assay. It may also be measured in a 51-chromiumrelease assay. It is represented by the EC₅₀ value, which corresponds tothe half maximal effective concentration (concentration of the antibodyconstruct which induces a cytotoxic response halfway between thebaseline and maximum). Preferably, the EC₅₀ value of the EGFRVII×CD3bispecific antibody constructs is ≤5000 pM or ≤4000 pM, more preferably≤3000 pM or ≤2000 pM, even more preferably ≤1000 pM or ≤500 pM, evenmore preferably ≤400 pM or ≤300 pM, even more preferably ≤200 pM, evenmore preferably ≤100 pM, even more preferably ≤50 pM, even morepreferably ≤20 pM or ≤10 pM, and most preferably ≤5 pM.

The above given EC₅₀ values can be measured in different assays. Theskilled person is aware that an EC₅₀ value can be expected to be lowerwhen stimulated I enriched CD8+ T cells are used as effector cells,compared with unstimulated PBMC. It can furthermore be expected that theEC₅₀ values are lower when the target cells express a high number of thetarget antigen compared with a low target expression rat. For example,when stimulated I enriched human CD8+ T cells are used as effector cells(and either EGFRVIII transfected cells such as CHO cells or a EGFRVIIIpositive human glioblastoma cell line U87 OR DK-MG are used as targetcells), the EC₅₀ value of the EGFRVIII ×CD3 bispecific antibodyconstruct is preferably ≤1000 pM, more preferably ≤500 pM, even morepreferably ≤250 pM, even more preferably ≤100 pM, even more preferably≤50 pM, even more preferably ≤10 pM, and most preferably ≤5 pM. Whenhuman PBMCs are used as effector cells, the EC₅₀ value of theEGFRVII×CD3 bispecific antibody construct is preferably ≤5000 pM or≤4000 pM (in particular when the target cells are a EGFRVIII positivehuman glioblastoma cell line U87 OR DK-MG), more preferably ≤2000 pM (inparticular when the target cells are EGFRVIII transfected cells such asCHO cells), more preferably ≤1000 pM or ≤500 pM, even more preferably≤200 pM, even more preferably ≤150 pM, even more preferably ≤100 pM, andmost preferably ≤50 pM, or lower. When a macaque T cell line such asLnPx4119 is used as effector cells, and a macaque EGFRVIII transfectedcell line such as CHO cells is used as target cell line, the EC₅₀ valueof the EGFRVIII ×CD3 bispecific antibody construct is preferably ≤2000pM or ≤1500 pM, more preferably ≤1000 pM or ≤500 pM, even morepreferably ≤300 pM or ≤250 pM, even more preferably ≤100 pM, and mostpreferably ≤50 pM.

Preferably, the EGFRVII×CD3 bispecific antibody constructs of thepresent invention do not induce/mediate lysis or do not essentiallyinduce/mediate lysis of EGFRVIII negative cells such as CHO cells. Theterm “do not induce lysis”, “do not essentially induce lysis”, “do notmediate lysis” or “do not essentially mediate lysis” means that anantibody construct of the present invention does not induce or mediatelysis of more than 30%, preferably not more than 20%, more preferablynot more than 10%, particularly preferably not more than 9%, 8%, 7%, 6%or 5% of EGFRVIII negative cells, whereby lysis of a EGFRVIII positivehuman glioblastoma cell line U87 OR DK-MG (see above) is set to be 100%.This usually applies for concentrations of the antibody construct of upto 500 nM. The skilled person knows how to measure cell lysis withoutfurther ado. Moreover, the present specification teaches specificinstructions how to measure cell lysis.

The difference in cytotoxic activity between the monomeric and thedimeric isoform of individual EGFRVIII ×CD3 bispecific antibodyconstructs is referred to as “potency gap”. This potency gap can e.g. becalculated as ratio between EC₅₀ values of the molecule's monomeric anddimeric form, see Example 1.8. Potency gaps of the EGFRVII×CD3bispecific antibody constructs of the present invention are preferably≤5, more preferably ≤4, even more preferably ≤3, even more preferably≤2, furthermore preferably ≤1, and most preferably ≤0.3.

The first and/or the second (or any further) binding domain(s) of theantibody construct of the invention is/are preferably cross-speciesspecific for members of the mammalian order of primates. Cross-speciesspecific CD3 binding domains are, for example, described in WO2008/119567. According to one embodiment, the first and/or secondbinding domain, in addition to binding to human EGFRVIII and human CD3,respectively, will also bind to EGFRVIII/CD3 of primates including (butnot limited to) new world primates (such as Callithrix jacchus, SaguinusOedipus or Saimiri sciureus), old world primates (such baboons andmacaques), gibbons, orangutans, and non-human homininae. It is envisagedthat the first binding domain of the antibody construct of the inventionwhich binds to human EGFRVIII on the surface of a target cell also bindsat least to macaque EGFRVIII, and/or the second binding domain whichbinds to human CD3 on the surface of a T cell also binds at least tomacaque CD3. A preferred macaque is Macaca fascicularis. Macaca mulatta(Rhesus) is also envisaged.

In one aspect of the invention, the first binding domain binds to humanEGFRVIII and further binds to macaque EGFRVIII, such as EGFRVIII ofMacaca fascicularis, and more preferably, to macaque EGFRVIII expressedon the surface macaque cells. A preferred Macaca fascicularis EGFRVIIIis depicted in SEQ ID NO: 234. The affinity of the first binding domainfor macaque EGFRVIII is preferably ≤15 nM, more preferably ≤10 nM, evenmore preferably ≤5 nM, even more preferably ≤1 nM, even more preferably≤0.5 nM, even more preferably ≤0.1 nM, and most preferably 50.05 nM oreven 50.01 nM.

Preferably the affinity gap of the antibody constructs according to theinvention for binding macaque EGFRVIII versus human EGFRVIII [maEGFRVIII:hu EGFRVIII] (as determined e.g. by BiaCore or by Scatchardanalysis) is <100, preferably <20, more preferably <15, furtherpreferably <10, even more preferably <8, more preferably <6 and mostpreferably <2. Preferred ranges for the affinity gap of the antibodyconstructs according to the invention for binding macaque EGFRVIIIversus human EGFRVIII are between 0.1 and 20, more preferably between0.2 and 10, even more preferably between 0.3 and 6, even more preferablybetween 0.5 and 3 or between 0.5 and 2.5, and most preferably between0.5 and 2 or between 0.6 and 2.

In one embodiment of the antibody construct of the invention, the secondbinding domain binds to human CD3 epsilon and to Callithrix jacchus,Saguinus Oedipus or Saimiri sciureus CD3 epsilon. Preferably, the secondbinding domain binds to an extracellular epitope of these CD3 epsilonchains. It is also envisaged that the second binding domain binds to anextracellular epitope of the human and the Macaca CD3 epsilon chain. Themost preferred epitope of CD3 epsilon is comprised within amino acidresidues 1-27 of the human CD3 epsilon extracellular domain. Even morespecifically, the epitope comprises at least the amino acid sequenceGln-Asp-Gly-Asn-Glu (SEQ ID NO: 169). Callithrix jacchus and Saguinusoedipus are both new world primate belonging to the family ofCallitrichidae, while Saimiri sciureus is a new world primate belongingto the family of Cebidae.

It is particularly preferred for the antibody construct of the presentinvention that the second binding domain which binds to human CD3 on thesurface of a T cell comprises a VL region comprising CDR-L1, CDR-L2 andCDR-L3 selected from:

-   (a) CDR-L1 as depicted in SEQ ID NO: 27 of WO 2008/119567, CDR-L2 as    depicted in SEQ ID NO: 28 of WO 2008/119567 and CDR-L3 as depicted    in SEQ ID NO: 29 of WO 2008/119567;-   (b) CDR-L1 as depicted in SEQ ID NO: 117 of WO 2008/119567, CDR-L2    as depicted in SEQ ID NO: 118 of WO 2008/119567 and CDR-L3 as    depicted in SEQ ID NO: 119 of WO 2008/119567; and-   (c) CDR-L1 as depicted in SEQ ID NO: 153 of WO 2008/119567, CDR-L2    as depicted in SEQ ID NO: 154 of WO 2008/119567 and CDR-L3 as    depicted in SEQ ID NO: 155 of WO 2008/119567.

In an alternatively preferred embodiment of the antibody construct ofthe present invention, the second binding domain which binds to humanCD3 on the surface of a T cell comprises a VH region comprising CDR-H 1,CDR-H2 and CDR-H3 selected from:

-   (a) CDR-H1 as depicted in SEQ ID NO: 12 of WO 2008/119567, CDR-H2 as    depicted in SEQ ID NO: 13 of WO 2008/119567 and CDR-H3 as depicted    in SEQ ID NO: 14 of WO 2008/119567;-   (b) CDR-H1 as depicted in SEQ ID NO: 30 of WO 2008/119567, CDR-H2 as    depicted in SEQ ID NO: 31 of WO 2008/119567 and CDR-H3 as depicted    in SEQ ID NO: 32 of WO 2008/119567;-   (c) CDR-H1 as depicted in SEQ ID NO: 48 of WO 2008/119567, CDR-H2 as    depicted in SEQ ID NO: 49 of WO 2008/119567 and CDR-H3 as depicted    in SEQ ID NO: 50 of WO 2008/119567;-   (d) CDR-H1 as depicted in SEQ ID NO: 66 of WO 2008/119567, CDR-H2 as    depicted in SEQ ID NO: 67 of WO 2008/119567 and CDR-H3 as depicted    in SEQ ID NO: 68 of WO 2008/119567;-   (e) CDR-H1 as depicted in SEQ ID NO: 84 of WO 2008/119567, CDR-H2 as    depicted in SEQ ID NO: 85 of WO 2008/119567 and CDR-H3 as depicted    in SEQ ID NO: 86 of WO 2008/119567;-   (f) CDR-H1 as depicted in SEQ ID NO: 102 of WO 2008/119567, CDR-H2    as depicted in SEQ ID NO: 103 of WO 2008/119567 and CDR-H3 as    depicted in SEQ ID NO: 104 of WO 2008/119567;-   (g) CDR-H1 as depicted in SEQ ID NO: 120 of WO 2008/119567, CDR-H2    as depicted in SEQ ID NO: 121 of WO 2008/119567 and CDR-H3 as    depicted in SEQ ID NO: 122 of WO 2008/119567;-   (h) CDR-H1 as depicted in SEQ ID NO: 138 of WO 2008/119567, CDR-H2    as depicted in SEQ ID NO: 139 of WO 2008/119567 and CDR-H3 as    depicted in SEQ ID NO: 140 of WO 2008/119567;-   (i) CDR-H1 as depicted in SEQ ID NO: 156 of WO 2008/119567, CDR-H2    as depicted in SEQ ID NO: 157 of WO 2008/119567 and CDR-H3 as    depicted in SEQ ID NO: 158 of WO 2008/119567; and-   (j) CDR-H1 as depicted in SEQ ID NO: 174 of WO 2008/119567, CDR-H2    as depicted in SEQ ID NO: 175 of WO 2008/119567 and CDR-H3 as    depicted in SEQ ID NO: 176 of WO 2008/119567.

It is further preferred for the antibody construct of the presentinvention that the second binding domain which binds to human CD3 on thesurface of a T cell comprises a VL region selected from the groupconsisting of a VL region as depicted in SEQ ID NO: 18, SEQ ID NO: 27,SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO:72, SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, and SEQ ID NO: 102 (seealso SEQ ID NO: 35, 39, 125, 129, 161 or 165 of WO 2008/119567).

It is alternatively preferred that the second binding domain which bindsto human CD3 on the surface of a T cell comprises a VH region selectedfrom the group consisting of a VH region as depicted in SEQ ID NO: 17,SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO:62, SEQ ID NO: 71, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98, and SEQID NO: 101 (see also SEQ ID NO: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91,105, 109, 123, 127, 141, 145, 159, 163, 177 or 181 of WO 2008/119567).

More preferably, the antibody construct of the present invention ischaracterized by the second binding domain which binds to human CD3 onthe surface of a T cell comprising a VL region and a VH region selectedfrom the group consisting of:

-   (a) a VL region as depicted in SEQ ID NO: 17 or 21 of WO 2008/119567    and a VH region as depicted in SEQ ID NO: 15 or 19 of WO    2008/119567;-   (b) a VL region as depicted in SEQ ID NO: 35 or 39 of WO 2008/119567    and a VH region as depicted in SEQ ID NO: 33 or 37 of WO    2008/119567;-   (c) a VL region as depicted in SEQ ID NO: 53 or 57 of WO 2008/119567    and a VH region as depicted in SEQ ID NO: 51 or 55 of WO    2008/119567;-   (d) a VL region as depicted in SEQ ID NO: 71 or 75 of WO 2008/119567    and a VH region as depicted in SEQ ID NO: 69 or 73 of WO    2008/119567;-   (e) a VL region as depicted in SEQ ID NO: 89 or 93 of WO 2008/119567    and a VH region as depicted in SEQ ID NO: 87 or 91 of WO    2008/119567;-   (f) a VL region as depicted in SEQ ID NO: 107 or 111 of WO    2008/119567 and a VH region as depicted in SEQ ID NO: 105 or 109 of    WO 2008/119567;-   (g) a VL region as depicted in SEQ ID NO: 125 or 129 of WO    2008/119567 and a VH region as depicted in SEQ ID NO: 123 or 127 of    WO 2008/119567;-   (h) a VL region as depicted in SEQ ID NO: 143 or 147 of WO    2008/119567 and a VH region as depicted in SEQ ID NO: 141 or 145 of    WO 2008/119567;-   (i) a VL region as depicted in SEQ ID NO: 161 or 165 of WO    2008/119567 and a VH region as depicted in SEQ ID NO: 159 or 163 of    WO 2008/119567; and-   (j) a VL region as depicted in SEQ ID NO: 179 or 183 of WO    2008/119567 and a VH region as depicted in SEQ ID NO: 177 or 181 of    WO 2008/119567.

Also preferred in connection with the antibody construct of the presentinvention is a second binding domain which binds to human CD3 on thesurface of a T cell comprising a VL region as depicted in SEQ ID NO: 102and a VH region as depicted in SEQ ID NO: 101.

According to a preferred embodiment of the antibody construct of thepresent invention, the binding domains and in particular the secondbinding domain (which binds to human CD3 on the surface of a T cell)have the following format: The pairs of VH regions and VL regions are inthe format of a single chain antibody (scFv). The VH and VL regions arearranged in the order VH-VL or VL-VH. It is preferred that the VH-regionis positioned N-terminally of a linker sequence, and the VL-region ispositioned C-terminally of the linker sequence.

A preferred embodiment of the above described antibody construct of thepresent invention is characterized by the second binding domain whichbinds to human CD3 on the surface of a T cell comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO:28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ IDNO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103(see also SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115,131, 133, 149, 151, 167, 169, 185 or 187 of WO 2008/119567).

It is also envisaged that the antibody construct of the invention has,in addition to its function to bind to the target molecules EGFRVIII andCD3, a further function. In this format, the antibody construct is atrifunctional or multifunctional antibody construct by targeting targetcells through binding to EGFRVIII, mediating cytotoxic T cell activitythrough CD3 binding and providing a further function such as a fullyfunctional Fc constant domain mediating antibody-dependent cellularcytotoxicity through recruitment of effector cells like NK cells, alabel (fluorescent etc.), a therapeutic agent such as a toxin orradionuclide, and/or means to enhance serum half-life, etc.

Examples for means to extend serum half-life of the antibody constructsof the invention include peptides, proteins or domains of proteins,which are fused or otherwise attached to the antibody constructs. Thegroup of peptides, proteins or protein domains includes peptides bindingto other proteins with preferred pharmacokinetic profile in the humanbody such as serum albumin (see WO 2009/127691). An alternative conceptof such half-life extending peptides includes peptides binding to theneonatal Fc receptor (FcRn, see WO 2007/098420), which can also be usedin the constructs of the present invention. The concept of attachinglarger domains of proteins or complete proteins includes e.g. the fusionof human serum albumin, variants or mutants of human serum albumin (seeWO 2011/051489, WO 2012/059486, WO 2012/150319, WO 2013/135896, WO2014/072481, WO 2013/075066) or domains thereof as well as the fusion ofconstant region of immunoglobulins (Fc domains) and variants thereof.Such variants of Fc domains may be optimized/modified in order to allowthe desired pairing of dimers or mulimers, to abolish Fc receptorbinding (e.g. the Fcγ receptor) or for other reasons. A further conceptknown in the art to extend the half-life of small protein compounds inthe human body is the pegylation of those compounds such as the antibodyconstruct of the present invention.

In a preferred embodiment, the antibody construct of the invention isdescribed as follows:

-   (a) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1-9; and    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;        and    -   optionally a His-tag, such as the one depicted in SEQ ID NO 10;-   (b) a polypeptide comprising in following order starting from the    N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1-9;    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;    -   optionally a peptide linker having an amino acid sequence        selected from the group consisting of SEQ ID NOs: 1-9;    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 104-134; and    -   optionally a His-tag, such as the one depicted in SEQ ID NO 10;-   (c) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having the amino acid sequence QRFVTGHFGGLX₁PANG        (SEQ ID NO: 135) whereas X₁ is Y or H; and    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1-9;    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;    -   a polypeptide having the amino acid sequence QRFVTGHFGGLHPANG        (SEQ ID NO: 137) or QRFCTGHFGGLHPCNG (SEQ ID NO: 139); and    -   optionally a His-tag, such as the one depicted in SEQ ID NO 10;-   (d) a polypeptide comprising in the following order starting from    the N-terminus    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35,        SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ        ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98, and SEQ ID NO: 101;    -   a peptide linker having the amino acid sequence depicted in SEQ        ID NO: 8;    -   a polypeptide having an amino acid of SEQ ID NO: 158;    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 140; and-    a polypeptide comprising in the following order starting from the    N-terminus:    -   a polypeptide having an amino acid sequence s of SEQ ID NO: 157;    -   a peptide linker having the amino acid sequence depicted in SEQ        ID NO: 8;    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36,        SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ        ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, and SEQ ID NO: 102 and        a serine residue at the C-terminus;    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 141;-   (e) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35,        SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ        ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98, and SEQ ID NO: 101;    -   a peptide linker having the amino acid sequence depicted in SEQ        ID NO: 8;    -   a polypeptide having an amino acid sequence of SEQ ID NO: 158;    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 142; and-    a polypeptide comprising in the following order starting from the    N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 157;    -   a peptide linker having an amino acid sequence depicted in SEQ        ID NO: 8;    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36,        SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ        ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, and SEQ ID NO: 102 and        a serine residue at the C-terminus;    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 143;-   (f) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1-9; and    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;        and    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 144; and a polypeptide having the amino acid sequence        depicted in SEQ ID NO: 145;-   (g) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;        and    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 146; and-    a polypeptide comprising in the following order starting from the    N-terminus:    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;        and    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 147;-   (h) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 148; and-    a polypeptide comprising in the following order starting from the    N-terminus:    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;        and    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 149; or-   (i) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1-9; and    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;        and    -   a polypeptide having the amino acid sequence depicted in SEQ ID        NO: 150.-   (j) a polypeptide comprising in the following order starting from    the N-terminus:    -   a polypeptide having an amino acid sequence of SEQ ID NO: 159;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1-9; and    -   a polypeptide having an amino acid sequence selected from the        group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37,        SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ        ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 8 and 9; and    -   the third domain having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 181-188.

As described above, several preferred antibody constructs of theinvention are modified by fusion with another moiety such as albumin oralbumin variants. If these fusion constructs are characterized for theirproperties, in particular target affinity or cytotoxic activity, theskilled person will be aware that similar fusion constructs orunmodified bispecific antibody constructs can be expected to havesimilar (or even better) properties. For example, if a bispecificantibody construct fused with albumin has an appreciable or desirablecytotoxic activity or target affinity, it can be expected that thesame/similar or even a higher cytotoxic activity/target affinity will beobserved for the construct w/o albumin.

According to another preferred embodiment, the bispecific antibodyconstruct of the invention comprises (in addition to the two bindingdomains) a third domain which comprises two polypeptide monomers, eachcomprising a hinge, a CH2 and a CH3 domain, wherein said twopolypeptides (or polypeptide monomers) are fused to each other via apeptide linker. Preferably, said third domain comprises in an N- toC-terminal order: hinge-CH2-CH3-linker-hinge-CH2-CH3. Preferred aminoacid sequences for said third domain are depicted in SEQ ID NOs:181-188. Each of said two polypeptide monomers preferably has an aminoacid sequence that is selected from the group consisting of SEQ ID NOs:173-180, or that is at least 90% identical to those sequences. Inanother preferred embodiment, the first and second binding domains ofthe bispecific antibody construct of the invention are fused to thethird domain via a peptide linker which is for example selected from thegroup consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9.

In line with the present invention, a “hinge” is an IgG hinge region.This region can be identified by analogy using the Kabat numbering, seeKabat positions 223-243. In line with the above, the minimal requirementfor a “hinge” are the amino acid residues corresponding to the IgG₁sequence stretch of D231 to P243 according to the Kabat numbering. Theterms CH2 and CH3 refer to the immunoglobulin heavy chain constantregions 2 and 3. These regions can as well be identified by analogyusing the Kabat numbering, see Kabat positions 244-360 for CH2 and Kabatpositions 361-478 for CH3. Is understood that there is some variationbetween the immunoglobulins in terms of their IgG₁ Fc region, IgG₂ Fcregion, IgG₃ Fc region, IgG₄ Fc region, IgM Fc region, IgA Fc region,IgD Fc region and IgE Fc region (see, e.g., Padlan, MolecularImmunology, 31(3), 169-217 (1993)). The term Fc monomer refers to thelast two heavy chain constant regions of IgA, IgD, and IgG, and the lastthree heavy chain constant regions of IgE and IgM. The Fc monomer canalso include the flexible hinge N-terminal to these domains. For IgA andIgM, the Fc monomer may include the J chain. For IgG, the Fc portioncomprises immunoglobulin domains CH2 and CH3 and the hinge between thefirst two domains and CH2. Although the boundaries of the Fc portion ofan immunoglobulin may vary, an example for a human IgG heavy chain Fcportion comprising a functional hinge, CH2 and CH3 domain can be definede.g. to comprise residues D231 (of the hinge domain) to P476 (of theC-terminus of the CH3 domain), or D231 to L476, respectively, for IgG₄,wherein the numbering is according to Kabat.

The antibody construct of the invention may hence comprise in an N- toC-terminal order:

-   -   (a) the first binding domain;    -   (b) a peptide linker having an amino acid sequence selected from        the group consisting of SEQ ID NOs: SEQ ID NOs: 1-9;    -   (c) the second binding domain;    -   (d) a peptide linker having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 8 and 9;    -   (e) the first polypeptide monomer of the third domain        (comprising a hinge, a CH2 and a CH3 domain);    -   (f) a peptide linker having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 192, 193, 194 and 195; and    -   (g) the second polypeptide monomer of the third domain        (comprising a hinge, a CH2 and a CH3 domain).

It is also preferred that the antibody construct of the inventioncomprises in an N- to C-terminal order:

-   -   the first binding domain having an amino acid sequence selected        from the group consisting of SEQ ID NO: 159;    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1-9;    -   the second binding domain having an amino acid sequence selected        from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ        ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID        NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID        NO: 103 (see also SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79,        95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187 of WO        2008/119567);    -   a peptide linker having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 1, 2, 4, 5, 6, 8 and 9; and    -   the third domain having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 181-188.

Hence, in a preferred embodiment, the antibody construct of the presentinvention comprises or consists of a polypeptide depicted in SEQ ID NOs:189 or 190.

In one embodiment of the antibody construct of the invention theantibody construct comprises or consists of a polypeptide as depicted inSEQ ID NO: 160.

Covalent modifications of the antibody constructs are also includedwithin the scope of this invention, and are generally, but not always,done post-translationally. For example, several types of covalentmodifications of the antibody construct are introduced into the moleculeby reacting specific amino acid residues of the antibody construct withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl andamino terminal residues are reacted with succinic or other carboxylicacid anhydrides. Derivatization with these agents has the effect ofreversing the charge of the lysinyl residues. Other suitable reagentsfor derivatizing alpha-amino-containing residues include imidoesterssuch as methyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking theantibody constructs of the present invention to a water-insolublesupport matrix or surface for use in a variety of methods. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates as describedin U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the antibody constructsincluded within the scope of this invention comprises altering theglycosylation pattern of the protein. As is known in the art,glycosylation patterns can depend on both the sequence of the protein(e.g., the presence or absence of particular glycosylation amino acidresidues, discussed below), or the host cell or organism in which theprotein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody construct isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the amino acid sequence of an antibody construct is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody construct is by chemical or enzymatic coupling of glycosides tothe protein. These procedures are advantageous in that they do notrequire production of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330, and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

Removal of carbohydrate moieties present on the starting antibodyconstruct may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Other modifications of the antibody construct are also contemplatedherein. For example, another type of covalent modification of theantibody construct comprises linking the antibody construct to variousnon-proteinaceous polymers, including, but not limited to, variouspolyols such as polyethylene glycol, polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, as is knownin the art, amino acid substitutions may be made in various positionswithin the antibody construct, e.g. in order to facilitate the additionof polymers such as PEG.

In some embodiments, the covalent modification of the antibodyconstructs of the invention comprises the addition of one or morelabels. The labelling group may be coupled to the antibody construct viaspacer arms of various lengths to reduce potential steric hindrance.Various methods for labelling proteins are known in the art and can beused in performing the present invention. The term “label” or “labellinggroup” refers to any detectable label. In general, labels fall into avariety of classes, depending on the assay in which they are to bedetected—the following examples include, but are not limited to:

-   -   a) isotopic labels, which may be radioactive or heavy isotopes,        such as radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S,        ⁸⁹Zr, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I)    -   b) magnetic labels (e.g., magnetic particles)    -   c) redox active moieties    -   d) optical dyes (including, but not limited to, chromophores,        phosphors and fluorophores) such as fluorescent groups (e.g.,        FITC, rhodamine, lanthanide phosphors), chemiluminescent groups,        and fluorophores which can be either “small molecule” fluores or        proteinaceous fluores    -   e) enzymatic groups (e.g. horseradish peroxidase,        β-galactosidase, luciferase, alkaline phosphatase)    -   f) biotinylated groups    -   g) predetermined polypeptide epitopes recognized by a secondary        reporter (e.g., leucine zipper pair sequences, binding sites for        secondary antibodies, metal binding domains, epitope tags, etc.)

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookby Richard P. Haugland.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), p galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658; 5,418,155;5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995;5,925,558).

Leucine zipper domains are peptides that promote oligomerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., 1988,Science 240:1759), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble oligomericproteins are described in PCT application WO 94/10308, and the leucinezipper derived from lung surfactant protein D (SPD) described in Hoppeet al., 1994, FEBS Letters 344:191. The use of a modified leucine zipperthat allows for stable trimerization of a heterologous protein fusedthereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78.In one approach, recombinant fusion proteins comprising a EGFRVIIIantibody fragment or derivative fused to a leucine zipper peptide areexpressed in suitable host cells, and the soluble oligomeric EGFRVIIIantibody fragments or derivatives that form are recovered from theculture supernatant.

The antibody construct of the invention may also comprise additionaldomains, which are e.g. helpful in the isolation of the molecule orrelate to an adapted pharmacokinetic profile of the molecule. Domainshelpful for the isolation of an antibody construct may be selected frompeptide motives or secondarily introduced moieties, which can becaptured in an isolation method, e.g. an isolation column. Non-limitingembodiments of such additional domains comprise peptide motives known asMyc-tag, HAT-tag, HA-tag, TAP-tag, GST-tag, chitin binding domain(CBD-tag), maltose binding protein (MBP-tag), Flag-tag, Strep-tag andvariants thereof (e.g. Strepll-tag) and His-tag. All herein disclosedantibody constructs characterized by the identified CDRs are preferredto comprise a His-tag domain, which is generally known as a repeat ofconsecutive His residues in the amino acid sequence of a molecule,preferably of five, and more preferably of six His residues(hexa-histidine). The His-tag may be located e.g. at the N- orC-terminus of the antibody construct, preferably it is located at theC-terminus. Most preferably, a hexa-histidine tag (HHHHHH) (SEQ IDNO:10) is linked via peptide bond to the C-terminus of the antibodyconstruct according to the invention.

The first binding domain of the antibody construct of the presentinvention binds to human EGFRVIII on the surface of a target cell. Thepreferred amino acid sequence of human EGFRVIII is represented by NOs:231, 232, and 233. The first binding domain according to the inventionhence preferably binds to EGFRVIII when it is expressed by naturallyexpressing cells or cell lines, and/or by cells or cell linestransformed or (stably/transiently) transfected with EGFRVIII. In apreferred embodiment the first binding domain also binds to EGFRVIIIwhen EGFRVIII is used as a “target” or “ligand” molecule in an in vitrobinding assay such as BIAcore or Scatchard. The “target cell” can be anyprokaryotic or eukaryotic cell expressing EGFRVIII on its surface;preferably the target cell is a cell that is part of the human or animalbody, such as a ovarian cancer cell, pancreatic cancer cell,mesothelioma cell, lung cancer cell, gastric cancer cell and triplenegative breast cancer cell.

The affinity of the first binding domain for human EGFRVIII ispreferably ≤20 nM, more preferably ≤10 nM, even more preferably ≤5 nM,even more preferably ≤2 nM, even more preferably ≤1 nM, even morepreferably ≤0.6 nM, even more preferably ≤0.5 nM, and most preferably≤0.4 nM. The affinity can be measured for example in a BIAcore assay orin a Scatchard assay, e.g. as described in the Examples. Other methodsof determining the affinity are also well-known to the skilled person;see e.g. appended Examples.

Amino acid sequence modifications of the antibody constructs describedherein are also contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody construct. Amino acid sequence variants of the antibodyconstructs are prepared by introducing appropriate nucleotide changesinto the antibody constructs nucleic acid, or by peptide synthesis. Allof the below described amino acd sequence modifications should result inan antibody construct which still retains the desired biologicalactivity (binding to EGFRVIII and to CD3) of the unmodified parentalmolecule.

The term “amino acid” or “amino acid residue” typically refers to anamino acid having its art recognized definition such as an amino acidselected from the group consisting of: alanine (Ala or A); arginine (Argor R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys orC); glutamine (GIn or Q); glutamic acid (Glu or E); glycine (Gly or G);histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine(Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line(Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp orW); tyrosine (Tyr or Y); and valine (Val or V), although modified,synthetic, or rare amino acids may be used as desired. Generally, aminoacids can be grouped as having a nonpolar side chain (e.g., Ala, Cys,He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g.,Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or anuncharged polar side chain (e.g., Asn, Cys, Gin, Gly, His, Met, Phe,Ser, Thr, Trp, and Tyr).

Amino acid modifications include, for example, deletions from, and/orinsertions into, and/or substitutions of, residues within the amino acidsequences of the antibody constructs. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody constructs, such as changing the number or position ofglycosylation sites.

For example, 1, 2, 3, 4, 5, or 6 amino acids may be inserted or deletedin each of the CDRs (of course, dependent on their length), while 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25amino acids may be inserted or deleted in each of the FRs. Preferably,amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residuesto polypeptides containing a hundred or more residues, as well asintra-sequence insertions of single or multiple amino acid residues. Aninsertional variant of the antibody construct of the invention includesthe fusion to the N-terminus or to the C-terminus of the antibodyconstruct of an enzyme or the fusion to a polypeptide which increasesthe serum half-life of the antibody construct.

The sites of greatest interest for substitutional mutagenesis includethe CDRs of the heavy and/or light chain, in particular thehypervariable regions, but FR alterations in the heavy and/or lightchain are also contemplated. The substitutions are preferablyconservative substitutions as described herein. Preferably, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids may be substituted in a CDR, while 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or25 amino acids may be substituted in the framework regions (FRs),depending on the length of the CDR or FR. For example, if a CDR sequenceencompasses 6 amino acids, it is envisaged that one, two or three ofthese amino acids are substituted. Similarly, if a CDR sequenceencompasses 15 amino acids it is envisaged that one, two, three, four,five or six of these amino acids are substituted.

A useful method for identification of certain residues or regions of theantibody constructs that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244: 1081-1085 (1989). Here, a residue or group oftarget residues within the antibody construct is/are identified (e.g.charged residues such as arg, asp, his, lys, and glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with theepitope.

Those amino acid locations demonstrating functional sensitivity to thesubstitutions are then refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site or regionfor introducing an amino acid sequence variation is predetermined, thenature of the mutation per se needs not to be predetermined. Forexample, to analyze or optimize the performance of a mutation at a givensite, alanine scanning or random mutagenesis may be conducted at atarget codon or region, and the expressed antibody construct variantsare screened for the optimal combination of desired activity. Techniquesfor making substitution mutations at predetermined sites in the DNAhaving a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants is done usingassays of antigen binding activities, such as EGFRVIII or CD3 binding.

Generally, if amino acids are substituted in one or more or all of theCDRs of the heavy and/or light chain, it is preferred that thethen-obtained “substituted” sequence is at least 60% or 65%, morepreferably 70% or 75%, even more preferably 80% or 85%, and particularlypreferably 90% or 95% identical to the “original” CDR sequence. Thismeans that it is dependent of the length of the CDR to which degree itis identical to the “substituted” sequence. For example, a CDR having 5amino acids is preferably 80% identical to its substituted sequence inorder to have at least one amino acid substituted. Accordingly, the CDRsof the antibody construct may have different degrees of identity totheir substituted sequences, e.g., CDRL1 may have 80%, while CDRL3 mayhave 90%.

Preferred substitutions (or replacements) are conservativesubstitutions. However, any substitution (including non-conservativesubstitution or one or more from the “exemplary substitutions” listed inTable 1, below) is envisaged as long as the antibody construct retainsits capability to bind to EGFRVIII via the first binding domain and toCD3 or CD3 epsilon via the second binding domain and/or its CDRs have anidentity to the then substituted sequence (at least 60% or 65%, morepreferably 70% or 75%, even more preferably 80% or 85%, and particularlypreferably 90% or 95% identical to the “original” CDR sequence).

Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened for a desired characteristic.

TABLE 1 Amino acid substitutions Preferred Original ExemplarySubstitutions Substitutions Ala (A) val, leu, ile val Arg (R) lys, gln,asn lys Asn (N) gln, his, asp, lys, arg gln Asp (D) glu, asn glu Cys (C)ser, ala ser Gln (Q) asn, glu asn Glu (E) asp, gln asp Gly (G) Ala alaHis (H) asn, gln, lys, arg arg Ile (I) leu, val, met, ala, phe leu Leu(L) norleucine, ile, val, met, ala ile Lys (K) arg, gln, asn arg Met (M)leu, phe, ile leu Phe (F) leu, val, ile, ala, tyr tyr Pro (P) Ala alaSer (S) Thr thr Thr (T) Ser ser Trp (W) tyr, phe tyr Tyr (Y) trp, phe,thr, ser phe Val (V) ile, leu, met, phe, ala leu

Substantial modifications in the biological properties of the antibodyconstruct of the present invention are accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues are divided intogroups based on common side-chain properties: (1) hydrophobic:norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser,thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5)residues that influence chain orientation: gly, pro; and (6) aromatic:trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the antibody construct may besubstituted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant crosslinking. Conversely, cysteinebond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=ll. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;Xu set to 16, and Xg set to 40 for database search stage and to 67 forthe output stage of the algorithms. Gapped alignments are triggered by ascore corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs are at least 60% to the sequences depictedherein, and more typically with preferably increasing homologies oridentities of at least 65% or 70%, more preferably at least 75% or 80%,even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, and almost 100%. In a similar manner, “percent (%)nucleic acid sequence identity” with respect to the nucleic acidsequence of the binding proteins identified herein is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues in the coding sequence of theantibody construct. A specific method utilizes the BLASTN module ofWU-BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andthe nucleotide sequences depicted herein are at least 60%, and moretypically with preferably increasing homologies or identities of atleast 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%.Thus, a “variant CDR” is one with the specified homology, similarity, oridentity to the parent CDR of the invention, and shares biologicalfunction, including, but not limited to, at least 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity ofthe parent CDR.

In one embodiment, the percentage of identity to human germline of theantibody constructs according to the invention is ≥70% or ≥75%, morepreferably ≥80% or ≥85%, even more preferably ≥90%, and most preferably≥91%, ≥92%, ≥93%, ≥94%, ≥95% or even ≥96%. Identity to human antibodygermline gene products is thought to be an important feature to reducethe risk of therapeutic proteins to elicit an immune response againstthe drug in the patient during treatment. Hwang & Foote (“Immunogenicityof engineered antibodies”; Methods 36 (2005) 3-10) demonstrate that thereduction of non-human portions of drug antibody constructs leads to adecrease of risk to induce anti-drug antibodies in the patients duringtreatment. By comparing an exhaustive number of clinically evaluatedantibody drugs and the respective immunogenicity data, the trend isshown that humanization of the V-regions of antibodies makes the proteinless immunogenic (average 5.1% of patients) than antibodies carryingunaltered non-human V regions (average 23.59% of patients). A higherdegree of identity to human sequences is hence desirable for V-regionbased protein therapeutics in the form of antibody constructs. For thispurpose of determining the germline identity, the V-regions of VL can bealigned with the amino acid sequences of human germline V segments and Jsegments (http://vbase.mrc-cpe.cam.ac.uk/) using Vector NTI software andthe amino acid sequence calculated by dividing the identical amino acidresidues by the total number of amino acid residues of the VL inpercent. The same can be for the VH segments(http://vbase.mrc-cpe.cam.ac.uk/) with the exception that the VH CDR3may be excluded due to its high diversity and a lack of existing humangermline VH CDR3 alignment partners. Recombinant techniques can then beused to increase sequence identity to human antibody germline genes.

In a further embodiment, the bispecific antibody constructs of thepresent invention exhibit high monomer yields under standard researchscale conditions, e.g., in a standard two-step purification process.Preferably the monomer yield of the antibody constructs according to theinvention is ≥0.25 mg/L supernatant, more preferably ≥0.5 mg/L, evenmore preferably ≥1 mg/L, and most preferably ≥3 mg/L supernatant.

Likewise, the yield of the dimeric antibody construct isoforms and hencethe monomer percentage (i.e., monomer: (monomer+dimer)) of the antibodyconstructs can be determined. The productivity of monomeric and dimericantibody constructs and the calculated monomer percentage can e.g. beobtained in the SEC purification step of culture supernatant fromstandardized research-scale production in roller bottles. In oneembodiment, the monomer percentage of the antibody constructs is ≥80%,more preferably ≥85%, even more preferably ≥90%, and most preferably≥95%.

In one embodiment, the antibody constructs have a preferred plasmastability (ratio of EC50 with plasma to EC50 w/o plasma) of ≤5 or ≤4,more preferably ≤3.5 or ≤3, even more preferably ≤2.5 or ≤2, and mostpreferably ≤1.5 or ≤1. The plasma stability of an antibody construct canbe tested by incubation of the construct in human plasma at 37° C. for24 hours followed by EC50 determination in a 51-chromium releasecytotoxicity assay. The effector cells in the cytotoxicity assay can bestimulated enriched human CD8 positive T cells. Target cells can e.g. beCHO cells transfected with human EGFRVIII. The effector to target cell(E:T) ratio can be chosen as 10:1. The human plasma pool used for thispurpose is derived from the blood of healthy donors collected by EDTAcoated syringes. Cellular components are removed by centrifugation andthe upper plasma phase is collected and subsequently pooled. As control,antibody constructs are diluted immediately prior to the cytotoxicityassay in RPMI-1640 medium. The plasma stability is calculated as ratioof EC50 (after plasma incubation) to EC50 (control). See Example 7.

It is furthermore preferred that the monomer to dimer conversion ofantibody constructs of the invention is low. The conversion can bemeasured under different conditions and analyzed by high performancesize exclusion chromatography. See Example 5. For example, incubation ofthe monomeric isoforms of the antibody constructs can be carried out for7 days at 37° C. and concentrations of e 250 pg/ml in an incubator.Under these conditions, it is preferred that the antibody constructs ofthe invention show a dimer percentage that is ≤2.5%, more preferably≤2%, further preferred ≤1.5%, further preferably ≤1%, more prefereably≤0.5% and most prefereably ≤0.25%. While the Ev111-2 based bispecificantibody construct shows a dimer conversion rate of 1.56%, the EvIII-1based bispecific antibody construct of the invention shows a conversionrate at the upper boarder of the most preferred limit for thischaracteristic.

It is also preferred that the bispecific antibody constructs of thepresent invention present with very low dimer conversion after a numberof freeze/thaw cycles. For example, the antibody construct monomer isadjusted to a concentration of 250 pg/ml e.g. in generic formulationbuffer and subjected to three freeze/thaw cycles (freezing at −80° C.for 30 min followed by thawing for 30 min at room temperature), followedby high performance SEC to determine the percentage of initiallymonomeric antibody construct, which had been converted into dimericantibody construct. Preferably the dimer percentages of the bispecificantibody constructs are ≤2.5%, more preferably ≤2%, further preferred≤1.5%, further preferably ≤1%, and most prefereably ≤0.5%, for exampleafter three freeze/thaw cycles. While the EvIII-2 based bispecificantibody construct does not meet the preferred range (2.53% dimer), theEvIII-1 based bispecific antibody construct of the invention shows aconversion rate at the upper boarder of the most preferred limit forthis characteristic (0.59% dimer).

The bispecific antibody constructs of the present invention preferablyshow a favorable thermostability with aggregation temperatures ≥45° C.or ≥50° C., more preferably ≥51° C., ≥52° C., ≥53° C. or ≥54° C., evenmore preferably ≥56° C. or ≥57° C., and most preferably ≥58° C. or ≥59°C. The thermostability parameter can be determined in terms of antibodyaggregation temperature as follows: Antibody solution at a concentration250 pg/ml is transferred into a single use cuvette and placed in aDynamic Light Scattering (DLS) device. The sample is heated from 40° C.to 70° C. at a heating rate of 0.5° C./min with constant acquisition ofthe measured radius. Increase of radius indicating melting of theprotein and aggregation is used to calculate the aggregation temperatureof the antibody. See Example 6.

Alternatively, temperature melting curves can be determined byDifferential Scanning Calorimetry (DSC) to determine intrinsicbiophysical protein stabilities of the antibody constructs. Theseexperiments are performed using a MicroCal LLC (Northampton, Mass.,U.S.A) VP-DSC device. The energy uptake of a sample containing anantibody construct is recorded from 20° C. to 90° C. compared to asample containing only the formulation buffer. The antibody constructsare adjusted to a final concentration of 250 pg/ml e.g. in SEC runningbuffer. For recording of the respective melting curve, the overallsample temperature is increased stepwise. At each temperature T energyuptake of the sample and the formulation buffer reference is recorded.The difference in energy uptake Cp (kcal/mole/° C.) of the sample minusthe reference is plotted against the respective temperature. The meltingtemperature is defined as the temperature at the first maximum of energyuptake.

The EGFRVIII ×CD3 bispecific antibody constructs of the invention arealso envisaged to have a turbidity (as measured by OD340 afterconcentration of purified monomeric antibody construct to 2.5 mg/ml andover night incubation) of ≤0.2, preferably of ≤0.15, more preferably of≤0.12, even more preferably of ≤0.1 or even ≥0.09, and most preferablyof ≤0.08 or ≥0.07. See Example 7. While the bispecific antibodyconstruct EvIII-2 shows a rather high turbidity (as measured by OD340after concentration of purified monomeric antibody construct to 2.5mg/ml and over night incubation) of almost 3, only the EvIII-1 basedbispecifc antibody construct is clearly within the desired spectrum forprotein compounds feasible for pharmaceutical formulation; see Table 3in Example 7.

In a further embodiment the antibody construct according to theinvention is stable at acidic pH. The more tolerant the antibodyconstruct behaves at unphysiologic pH such as pH 5.5 (a pH which isrequired to run e.g. a cation exchange chromatography), the higher isthe recovery of the antibody construct eluted from an ion exchangecolumn relative to the total amount of loaded protein. Recovery of theantibody construct from an ion (e.g., cation) exchange column at pH 5.5is preferably ≥30%, more preferably ≥40%, more preferably ≥50%, evenmore preferably ≥60%, even more preferably ≥70%, even more preferably≥80%, even more preferably ≥90%, even more preferably ≥95%, and mostpreferably ≥99%.

It is furthermore envisaged that the bispecific antibody constructs ofthe present invention exhibit therapeutic efficacy or anti-tumoractivity. This can e.g. be assessed in a study as disclosed in thefollowing example of an advanced stage human tumor xenograft model:

On day 1 of the study, 5×10⁶ cells of a human EGFRVIII positive cancercell line (e.g. human glioblastoma cell line U87 OR DK-MG) aresubcutaneously injected in the right dorsal flank of female NOD/SCIDmice. When the mean tumor volume reaches about 100 mm³, in vitroexpanded human CD3 positive T cells are transplanted into the mice byinjection of about 2×10⁷ cells into the peritoneal cavity of theanimals. Mice of vehicle control group 1 do not receive effector cellsand are used as an untransplanted control for comparison with vehiclecontrol group 2 (receiving effector cells) to monitor the impact of Tcells alone on tumor growth. The antibody treatment starts when the meantumor volume reaches about 200 mm³. The mean tumor size of eachtreatment group on the day of treatment start should not bestatistically different from any other group (analysis of variance).Mice are treated with 0.5 mg/kg/day of a EGFRVIII ×CD3 bispecifcantibody construct by intravenous bolus injection for about 15 to 20days. Tumors are measured by caliper during the study and progressevaluated by intergroup comparison of tumor volumes (TV). The tumorgrowth inhibition T/C [%] is determined by calculating TV as T/C%=100×(median TV of analyzed group)/(median TV of control group 2).

The skilled person knows how to modify or adapt certain parameters ofthis study, such as the number of injected tumor cells, the site ofinjection, the number of transplanted human T cells, the amount ofbispecific antibody constructs to be administered, and the timelines,while still arriving at a meaningful and reproducible result.Preferably, the tumor growth inhibition T/C [%] is ≤70 or ≤60, morepreferably ≤50 or ≤40, even more preferably ≤30 or ≤20 and mostpreferably ≤10 or ≤5 or even ≤2.5.

The invention further provides a polynucleotide/nucleic acid moleculeencoding an antibody construct of the invention.

A polynucleotide is a biopolymer composed of 13 or more nucleotidemonomers covalently bonded in a chain. DNA (such as cDNA) and RNA (suchas mRNA) are examples of polynucleotides with distinct biologicalfunction. Nucleotides are organic molecules that serve as the monomersor subunits of nucleic acid molecules like DNA or RNA. The nucleic acidmolecule or polynucleotide can be double stranded and single stranded,linear and circular. It is preferably comprised in a vector which ispreferably comprised in a host cell. Said host cell is, e.g. aftertransformation or transfection with the vector or the polynucleotide ofthe invention, capable of expressing the antibody construct. For thatpurpose the polynucleotide or nucleic acid molecule is operativelylinked with control sequences.

The genetic code is the set of rules by which information encoded withingenetic material (nucleic acids) is translated into proteins. Biologicaldecoding in living cells is accomplished by the ribosome which linksamino acids in an order specified by mRNA, using tRNA molecules to carryamino acids and to read the mRNA three nucleotides at a time. The codedefines how sequences of these nucleotide triplets, called codons,specify which amino acid will be added next during protein synthesis.With some exceptions, a three-nucleotide codon in a nucleic acidsequence specifies a single amino acid. Because the vast majority ofgenes are encoded with exactly the same code, this particular code isoften referred to as the canonical or standard genetic code. While thegenetic code determines the protein sequence for a given coding region,other genomic regions can influence when and where these proteins areproduced.

Furthermore, the invention provides a vector comprising apolynucleotide/nucleic acid molecule of the invention.

A vector is a nucleic acid molecule used as a vehicle to transfer(foreign) genetic material into a cell. The term “vector”encompasses—but is not restricted to—plasmids, viruses, cosmids andartificial chromosomes. In general, engineered vectors comprise anorigin of replication, a multicloning site and a selectable marker. Thevector itself is generally a nucleotide sequence, commonly a DNAsequence, that comprises an insert (transgene) and a larger sequencethat serves as the “backbone” of the vector. Modern vectors mayencompass additional features besides the transgene insert and abackbone: promoter, genetic marker, antibiotic resistance, reportergene, targeting sequence, protein purification tag. Vectors calledexpression vectors (expression constructs) specifically are for theexpression of the transgene in the target cell, and generally havecontrol sequences.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Transfection” is the process of deliberately introducing nucleic acidmolecules or polynucleotides (including vectors) into target cells. Theterm is mostly used for non-viral methods in eukaryotic cells.Transduction is often used to describe virus-mediated transfer ofnucleic acid molecules or polynucleotides. Transfection of animal cellstypically involves opening transient pores or “holes” in the cellmembrane, to allow the uptake of material. Transfection can be carriedout using calcium phosphate, by electroporation, by cell squeezing or bymixing a cationic lipid with the material to produce liposomes, whichfuse with the cell membrane and deposit their cargo inside.

The term “transformation” is used to describe non-viral transfer ofnucleic acid molecules or polynucleotides (including vectors) intobacteria, and also into non-animal eukaryotic cells, including plantcells. Transformation is hence the genetic alteration of a bacterial ornon-animal eukaryotic cell resulting from the direct uptake through thecell membrane(s) from its surroundings and subsequent incorporation ofexogenous genetic material (nucleic acid molecules). Transformation canbe effected by artificial means. For transformation to happen, cells orbacteria must be in a state of competence, which might occur as atime-limited response to environmental conditions such as starvation andcell density.

Moreover, the invention provides a host cell transformed or transfectedwith the polynucleotide/nucleic acid molecule or with the vector of theinvention.

As used herein, the terms “host cell” or “recipient cell” are intendedto include any individual cell or cell culture that can be or has/havebeen recipients of vectors, exogenous nucleic acid molecules, andpolynucleotides encoding the antibody construct of the presentinvention; and/or recipients of the antibody construct itself. Theintroduction of the respective material into the cell is carried out byway of transformation, transfection and the like. The term “host cell”is also intended to include progeny or potential progeny of a singlecell. Because certain modifications may occur in succeeding generationsdue to either natural, accidental, or deliberate mutation or due toenvironmental influences, such progeny may not, in fact, be completelyidentical (in morphology or in genomic or total DNA complement) to theparent cell, but is still included within the scope of the term as usedherein. Suitable host cells include prokaryotic or eukaryotic cells, andalso include but are not limited to bacteria, yeast cells, fungi cells,plant cells, and animal cells such as insect cells and mammalian cells,e.g., murine, rat, macaque or human.

The antibody construct of the invention can be produced in bacteria.After expression, the antibody construct of the invention is isolatedfrom the E. coli cell paste in a soluble fraction and can be purifiedthrough, e.g., affinity chromatography and/or size exclusion. Finalpurification can be carried out similar to the process for purifyingantibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for the antibodyconstruct of the invention. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe, Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12424),K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC56500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K.marxianus; yarrowia (EP 402 226); Pichia pastoris (EP 183 070); Candida;Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such asNeurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodyconstruct of the invention are derived from multicellular organisms.Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruit fly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,Arabidopsis and tobacco can also be used as hosts. Cloning andexpression vectors useful in the production of proteins in plant cellculture are known to those of skill in the art. See e.g. Hiatt et al.,Nature (1989) 342: 76-78, Owen et al. (1992) Bio/Technology 10: 790-794,Artsaenko et al. (1995) The Plant J 8: 745-750, and Fecker et al. (1996)Plant Mol Biol 32: 979-986.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36: 59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251(1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2,1413 8065); mouse mammary tumor (MMT060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N. Y Acad. Sci.(1982) 383: 44-68); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

In a further embodiment the invention provides a process for theproduction of an antibody construct of the invention, said processcomprising culturing a host cell of the invention under conditionsallowing the expression of the antibody construct of the invention andrecovering the produced antibody construct from the culture.

As used herein, the term “culturing” refers to the in vitro maintenance,differentiation, growth, proliferation and/or propagation of cells undersuitable conditions in a medium. The term “expression” includes any stepinvolved in the production of an antibody construct of the inventionincluding, but not limited to, transcription, post-transcriptionalmodification, translation, post-translational modification, andsecretion.

When using recombinant techniques, the antibody construct can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the antibody construct is produced intracellularly,as a first step, the particulate debris, either host cells or lysedfragments, are removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation. Where the antibody is secreted into themedium, supernatants from such expression systems are generally firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody construct of the invention prepared from the host cells canbe recovered or purified using, for example, hydroxylapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography. Other techniques for protein purification such asfractionation on an ion-exchange column, ethanol precipitation, ReversePhase HPLC, chromatography on silica, chromatography on heparinSEPHAROSE™, chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromato-focusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered. Where the antibody construct of the invention comprises a CH3domain, the Bakerbond ABX resin (J.T. Baker, Phillipsburg, N.J.) isuseful for purification.

Affinity chromatography is a preferred purification technique. Thematrix to which the affinity ligand is attached is most often agarose,but other matrices are available. Mechanically stable matrices such ascontrolled pore glass or poly (styrenedivinyl) benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose.

Moreover, the invention provides a pharmaceutical composition comprisingan antibody construct of the invention or an antibody construct producedaccording to the process of the invention. It is preferred for thepharmaceutical composition of the invention that the homogeneity of theantibody construct is ≥80%, more preferably ≥81%, ≥82%, ≥83%, ≥84%, or≥85%, further preferably ≥86%, ≥87%, ≥88%, ≥89%, or ≥90%, still furtherpreferably, ≥91%, ≥92%, ≥93%, ≥94%, or ≥95% and most preferably ≥96%,≥97%, ≥98% or ≥99%.

As used herein, the term “pharmaceutical composition” relates to acomposition which is suitable for administration to a patient,preferably a human patient. The particularly preferred pharmaceuticalcomposition of this invention comprises one or a plurality of theantibody construct(s) of the invention, preferably in a therapeuticallyeffective amount. Preferably, the pharmaceutical composition furthercomprises suitable formulations of one or more (pharmaceuticallyeffective) carriers, stabilizers, excipients, diluents, solubilizers,surfactants, emulsifiers, preservatives and/or adjuvants. Acceptableconstituents of the composition are preferably nontoxic to recipients atthe dosages and concentrations employed. Pharmaceutical compositions ofthe invention include, but are not limited to, liquid, frozen, andlyophilized compositions.

The inventive compositions may comprise a pharmaceutically acceptablecarrier. In general, as used herein, “pharmaceutically acceptablecarrier” means any and all aqueous and non-aqueous solutions, sterilesolutions, solvents, buffers, e.g. phosphate buffered saline (PBS)solutions, water, suspensions, emulsions, such as oil/water emulsions,various types of wetting agents, liposomes, dispersion media andcoatings, which are compatible with pharmaceutical administration, inparticular with parenteral administration. The use of such media andagents in pharmaceutical compositions is well known in the art, and thecompositions comprising such carriers can be formulated by well-knownconventional methods.

Certain embodiments provide pharmaceutical compositions comprising theantibody construct of the invention and further one or more excipientssuch as those illustratively described in this section and elsewhereherein. Excipients can be used in the invention in this regard for awide variety of purposes, such as adjusting physical, chemical, orbiological properties of formulations, such as adjustment of viscosity,and or processes of the invention to improve effectiveness and or tostabilize such formulations and processes against degradation andspoilage due to, for instance, stresses that occur during manufacturing,shipping, storage, pre-use preparation, administration, and thereafter.

In certain embodiments, the pharmaceutical composition may containformulation materials for the purpose of modifying, maintaining orpreserving, e.g., the pH, osmolarity, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition (see, REMINGTON'SPHARMACEUTICAL SCIENCES, 18″ Edition, (A.R. Genrmo, ed.), 1990, MackPublishing Company). In such embodiments, suitable formulation materialsmay include, but are not limited to:

-   -   amino acids such as glycine, alanine, glutamine, asparagine,        threonine, proline, 2-phenylalanine, including charged amino        acids, preferably lysine, lysine acetate, arginine, glutamate        and/or histidine    -   antimicrobials such as antibacterial and antifungal agents    -   antioxidants such as ascorbic acid, methionine, sodium sulfite        or sodium hydrogen-sulfite;    -   buffers, buffer systems and buffering agents which are used to        maintain the composition at physiological pH or at a slightly        lower pH, typically within a pH range of from about 5 to about 8        or 9; examples of buffers are borate, bicarbonate, Tris-HCl,        citrates, phosphates or other organic acids, succinate,        phosphate, histidine and acetate; for example Tris buffer of        about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5;    -   non-aqueous solvents such as propylene glycol, polyethylene        glycol, vegetable oils such as olive oil, and injectable organic        esters such as ethyl oleate;    -   aqueous carriers including water, alcoholic/aqueous solutions,        emulsions or suspensions, including saline and buffered media;    -   biodegradable polymers such as polyesters;    -   bulking agents such as mannitol or glycine;    -   chelating agents such as ethylenediamine tetraacetic acid        (EDTA);    -   isotonic and absorption delaying agents;    -   complexing agents such as caffeine, polyvinylpyrrolidone,        beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin)    -   fillers;    -   monosaccharides; disaccharides; and other carbohydrates (such as        glucose, mannose or dextrins); carbohydrates may be non-reducing        sugars, preferably trehalose, sucrose, octasulfate, sorbitol or        xylitol;    -   (low molecular weight) proteins, polypeptides or proteinaceous        carriers such as human or bovine serum albumin, gelatin or        immunoglobulins, preferably of human origin;    -   coloring and flavouring agents;    -   sulfur containing reducing agents, such as glutathione, thioctic        acid, sodium thioglycolate, thioglycerol,        [alpha]-monothioglycerol, and sodium thio sulfate    -   diluting agents;    -   emulsifying agents;    -   hydrophilic polymers such as polyvinylpyrrolidone)    -   salt-forming counter-ions such as sodium;    -   preservatives such as antimicrobials, anti-oxidants, chelating        agents, inert gases and the like; examples are: benzalkonium        chloride, benzoic acid, salicylic acid, thimerosal, phenethyl        alcohol, methylparaben, propylparaben, chlorhexidine, sorbic        acid or hydrogen peroxide);    -   metal complexes such as Zn-protein complexes;    -   solvents and co-solvents (such as glycerin, propylene glycol or        polyethylene glycol);    -   sugars and sugar alcohols, such as trehalose, sucrose,        octasulfate, mannitol, sorbitol or xylitol stachyose, mannose,        sorbose, xylose, ribose, myoinisitose, galactose, lactitol,        ribitol, myoinisitol, galactitol, glycerol, cyclitols (e.g.,        inositol), polyethylene glycol; and polyhydric sugar alcohols;    -   suspending agents;    -   surfactants or wetting agents such as pluronics, PEG, sorbitan        esters, polysorbates such as polysorbate 20, polysorbate,        triton, tromethamine, lecithin, cholesterol, tyloxapal;        surfactants may be detergents, preferably with a molecular        weight of >1.2 KD and/or a polyether, preferably with a        molecular weight of >3 KD; non-limiting examples for preferred        detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween        85; non-limiting examples for preferred polyethers are PEG 3000,        PEG 3350, PEG 4000 and PEG 5000;    -   stability enhancing agents such as sucrose or sorbitol;    -   tonicity enhancing agents such as alkali metal halides,        preferably sodium or potassium chloride, mannitol sorbitol;    -   parenteral delivery vehicles including sodium chloride solution,        Ringer's dextrose, dextrose and sodium chloride, lactated        Ringer's, or fixed oils;    -   intravenous delivery vehicles including fluid and nutrient        replenishers, electrolyte replenishers (such as those based on        Ringer's dextrose).

It is evident to those skilled in the art that the differentconstituents of the pharmaceutical composition (e.g., those listedabove) can have different effects, for example, and amino acid can actas a buffer, a stabilizer and/or an antioxidant; mannitol can act as abulking agent and/or a tonicity enhancing agent; sodium chloride can actas delivery vehicle and/or tonicity enhancing agent; etc.

It is envisaged that the composition of the invention might comprise, inaddition to the polypeptide of the invention defined herein, furtherbiologically active agents, depending on the intended use of thecomposition. Such agents might be drugs acting on the gastro-intestinalsystem, drugs acting as cytostatica, drugs preventing hyperurikemia,drugs inhibiting immunoreactions (e.g. corticosteroids), drugsmodulating the inflammatory response, drugs acting on the circulatorysystem and/or agents such as cytokines known in the art. It is alsoenvisaged that the antibody construct of the present invention isapplied in a co-therapy, i.e., in combination with another anti-cancermedicament.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibody construct of the invention. In certain embodiments, the primaryvehicle or carrier in a pharmaceutical composition may be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier maybe water for injection, physiological saline solution or artificialcerebrospinal fluid, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles. Incertain embodiments, the antibody construct of the inventioncompositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, the antibody construct of the invention may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired antibody construct of the invention in a pharmaceuticallyacceptable vehicle. A particularly suitable vehicle for parenteralinjection is sterile distilled water in which the antibody construct ofthe invention is formulated as a sterile, isotonic solution, properlypreserved. In certain embodiments, the preparation can involve theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that mayprovide controlled or sustained release of the product which can bedelivered via depot injection. In certain embodiments, hyaluronic acidmay also be used, having the effect of promoting sustained duration inthe circulation. In certain embodiments, implantable drug deliverydevices may be used to introduce the desired antibody construct.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving the antibody construct ofthe invention in sustained- or controlled-delivery/release formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, International PatentApplication No. PCT/US93/00829, which describes controlled release ofporous polymeric microparticles for delivery of pharmaceuticalcompositions. Sustained-release preparations may include semipermeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Sustained release matrices may include polyesters,hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 andEuropean Patent Application Publication No. EP 058481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers 2:547-556), poly (2-hydroxyethyl-methacrylate) (Langer etal., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem.Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may also includeliposomes that can be prepared by any of several methods known in theart. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949.

The antibody construct may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Another aspect of the invention includes self-buffering antibodyconstruct of the invention formulations, which can be used aspharmaceutical compositions, as described in international patentapplication WO 06138181A2 (PCT/US2006/022599). A variety of expositionsare available on protein stabilization and formulation materials andmethods useful in this regard, such as Arakawa et al., “Solventinteractions in pharmaceutical formulations,” Pharm Res. 8(3): 285-91(1991); Kendrick et al., “Physical stabilization of proteins in aqueoussolution” in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY ANDPRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002), and Randolph et al., “Surfactant-protein interactions”,Pharm Biotechnol. 13: 159-75 (2002), see particularly the partspertinent to excipients and processes of the same for self-bufferingprotein formulations in accordance with the current invention,especially as to protein pharmaceutical products and processes forveterinary and/or human medical uses.

Salts may be used in accordance with certain embodiments of theinvention to, for example, adjust the ionic strength and/or theisotonicity of a formulation and/or to improve the solubility and/orphysical stability of a protein or other ingredient of a composition inaccordance with the invention. As is well known, ions can stabilize thenative state of proteins by binding to charged residues on the protein'ssurface and by shielding charged and polar groups in the protein andreducing the strength of their electrostatic interactions, attractive,and repulsive interactions. Ions also can stabilize the denatured stateof a protein by binding to, in particular, the denatured peptidelinkages (—CONH) of the protein. Furthermore, ionic interaction withcharged and polar groups in a protein also can reduce intermolecularelectrostatic interactions and, thereby, prevent or reduce proteinaggregation and insolubility.

Ionic species differ significantly in their effects on proteins. Anumber of categorical rankings of ions and their effects on proteinshave been developed that can be used in formulating pharmaceuticalcompositions in accordance with the invention. One example is theHofmeister series, which ranks ionic and polar non-ionic solutes bytheir effect on the conformational stability of proteins in solution.Stabilizing solutes are referred to as “kosmotropic”. Destabilizingsolutes are referred to as “chaotropic”. Kosmotropes commonly are usedat high concentrations (e.g., >1 molar ammonium sulfate) to precipitateproteins from solution (“salting-out”). Chaotropes commonly are used todenture and/or to solubilize proteins (“salting-in”). The relativeeffectiveness of ions to “salt-in” and “salt-out” defines their positionin the Hofmeister series.

Free amino acids can be used in the antibody construct of the inventionformulations in accordance with various embodiments of the invention asbulking agents, stabilizers, and antioxidants, as well as other standarduses. Lysine, proline, serine, and alanine can be used for stabilizingproteins in a formulation. Glycine is useful in lyophilization to ensurecorrect cake structure and properties. Arginine may be useful to inhibitprotein aggregation, in both liquid and lyophilized formulations.Methionine is useful as an antioxidant.

Polyols include sugars, e.g., mannitol, sucrose, and sorbitol andpolyhydric alcohols such as, for instance, glycerol and propyleneglycol, and, for purposes of discussion herein, polyethylene glycol(PEG) and related substances. Polyols are kosmotropic. They are usefulstabilizing agents in both liquid and lyophilized formulations toprotect proteins from physical and chemical degradation processes.Polyols also are useful for adjusting the tonicity of formulations.Among polyols useful in select embodiments of the invention is mannitol,commonly used to ensure structural stability of the cake in lyophilizedformulations. It ensures structural stability to the cake. It isgenerally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucroseare among preferred agents for adjusting tonicity and as stabilizers toprotect against freeze-thaw stresses during transport or the preparationof bulks during the manufacturing process. Reducing sugars (whichcontain free aldehyde or ketone groups), such as glucose and lactose,can glycate surface lysine and arginine residues. Therefore, theygenerally are not among preferred polyols for use in accordance with theinvention. In addition, sugars that form such reactive species, such assucrose, which is hydrolyzed to fructose and glucose under acidicconditions, and consequently engenders glycation, also is not amongpreferred polyols of the invention in this regard. PEG is useful tostabilize proteins and as a cryoprotectant and can be used in theinvention in this regard.

Embodiments of the antibody construct of the invention formulationsfurther comprise surfactants. Protein molecules may be susceptible toadsorption on surfaces and to denaturation and consequent aggregation atair-liquid, solid-liquid, and liquid-liquid interfaces. These effectsgenerally scale inversely with protein concentration. These deleteriousinteractions generally scale inversely with protein concentration andtypically are exacerbated by physical agitation, such as that generatedduring the shipping and handling of a product. Surfactants routinely areused to prevent, minimize, or reduce surface adsorption. Usefulsurfactants in the invention in this regard include polysorbate 20,polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, andpoloxamer 188. Surfactants also are commonly used to control proteinconformational stability. The use of surfactants in this regard isprotein-specific since, any given surfactant typically will stabilizesome proteins and destabilize others.

Polysorbates are susceptible to oxidative degradation and often, assupplied, contain sufficient quantities of peroxides to cause oxidationof protein residue side-chains, especially methionine. Consequently,polysorbates should be used carefully, and when used, should be employedat their lowest effective concentration. In this regard, polysorbatesexemplify the general rule that excipients should be used in theirlowest effective concentrations.

Embodiments of the antibody construct of the invention formulationsfurther comprise one or more antioxidants. To some extent deleteriousoxidation of proteins can be prevented in pharmaceutical formulations bymaintaining proper levels of ambient oxygen and temperature and byavoiding exposure to light. Antioxidant excipients can be used as wellto prevent oxidative degradation of proteins. Among useful antioxidantsin this regard are reducing agents, oxygen/free-radical scavengers, andchelating agents. Antioxidants for use in therapeutic proteinformulations in accordance with the invention preferably arewater-soluble and maintain their activity throughout the shelf life of aproduct. EDTA is a preferred antioxidant in accordance with theinvention in this regard. Antioxidants can damage proteins. Forinstance, reducing agents, such as glutathione in particular, candisrupt intramolecular disulfide linkages. Thus, antioxidants for use inthe invention are selected to, among other things, eliminate orsufficiently reduce the possibility of themselves damaging proteins inthe formulation.

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins. Magnesium ions (10-120 mM) can be usedto inhibit isomerization of aspartic acid to isoaspartic acid. Ca⁺² ions(up to 100 mM) can increase the stability of human deoxyribonuclease.Mg⁺², Mn⁺², and Zn⁺², however, can destabilize rhDNase. Similarly, Ca⁺²and Sr⁺² can stabilize Factor VIII, it can be destabilized by Mg⁺², Mn⁺²and Zn⁺², Cu⁺² and Fe⁺², and its aggregation can be increased by Al⁺³ions.

Embodiments of the antibody construct of the invention formulationsfurther comprise one or more preservatives. Preservatives are necessarywhen developing multi-dose parenteral formulations that involve morethan one extraction from the same container. Their primary function isto inhibit microbial growth and ensure product sterility throughout theshelf-life or term of use of the drug product. Commonly usedpreservatives include benzyl alcohol, phenol and m-cresol. Althoughpreservatives have a long history of use with small-moleculeparenterals, the development of protein formulations that includespreservatives can be challenging. Preservatives almost always have adestabilizing effect (aggregation) on proteins, and this has become amajor factor in limiting their use in multi-dose protein formulations.To date, most protein drugs have been formulated for single-use only.However, when multi-dose formulations are possible, they have the addedadvantage of enabling patient convenience, and increased marketability.A good example is that of human growth hormone (hGH) where thedevelopment of preserved formulations has led to commercialization ofmore convenient, multi-use injection pen presentations. At least foursuch pen devices containing preserved formulations of hGH are currentlyavailable on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ(liquid, Genentech) & Genotropin (lyophilized—dual chamber cartridge,Pharmacia & Upjohn) contain phenol while Somatrope (Eli Lilly) isformulated with m-cresol. Several aspects need to be considered duringthe formulation and development of preserved dosage forms. The effectivepreservative concentration in the drug product must be optimized. Thisrequires testing a given preservative in the dosage form withconcentration ranges that confer anti-microbial effectiveness withoutcompromising protein stability.

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein, significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability should bemaintained over the entire product shelf-life (about 18 to 24 months).An important point to note is that preservative effectiveness should bedemonstrated in the final formulation containing the active drug and allexcipient components.

The antibody constructs disclosed herein may also be formulated asimmuno-liposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyconstruct are prepared by methods known in the art, such as described inEpstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang etal., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); U.S. Pat. Nos.4,485,045 and 4,544,545; and WO 97/38731. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Particularlyuseful liposomes can be generated by the reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. Fab′ fragments of the antibodyconstruct of the present invention can be conjugated to the liposomes asdescribed in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via adisulfide interchange reaction. A chemotherapeutic agent is optionallycontained within the liposome. See Gabizon et al. J. National CancerInst. 81 (19) 1484 (1989).

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration.

The biological activity of the pharmaceutical composition defined hereincan be determined for instance by cytotoxicity assays, as described inthe following examples, in WO 99/54440 or by Schlereth et al. (CancerImmunol. Immunother. 20 (2005), 1-12). “Efficacy” or “in vivo efficacy”as used herein refers to the response to therapy by the pharmaceuticalcomposition of the invention, using e.g. standardized NCI responsecriteria. The success or in vivo efficacy of the therapy using apharmaceutical composition of the invention refers to the effectivenessof the composition for its intended purpose, i.e. the ability of thecomposition to cause its desired effect, i.e. depletion of pathologiccells, e.g. tumor cells. The in vivo efficacy may be monitored byestablished standard methods for the respective disease entitiesincluding, but not limited to white blood cell counts, differentials,Fluorescence Activated Cell Sorting, bone marrow aspiration. Inaddition, various disease specific clinical chemistry parameters andother established standard methods may be used. Furthermore,computer-aided tomography, X-ray, nuclear magnetic resonance tomography(e.g. for National Cancer Institute-criteria based response assessment[Cheson B D, Horning S J, Coiffier B, Shipp M A, Fisher R I, Connors JM, Lister T A, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F,Klippensten D, Hiddemann W, Castellino R, Harris N L, Armitage J O,Carter W, Hoppe R, Canellos G P. Report of an international workshop tostandardize response criteria for non-Hodgkin's lymphomas. NCI SponsoredInternational Working Group. J Clin Oncol. 1999 April; 17(4):1244]),positron-emission tomography scanning, white blood cell counts,differentials, Fluorescence Activated Cell Sorting, bone marrowaspiration, lymph node biopsies/histologies, and various lymphomaspecific clinical chemistry parameters (e.g. lactate dehydrogenase) andother established standard methods may be used.

Another major challenge in the development of drugs such as thepharmaceutical composition of the invention is the predictablemodulation of pharmacokinetic properties. To this end, a pharmacokineticprofile of the drug candidate, i.e. a profile of the pharmacokineticparameters that affect the ability of a particular drug to treat a givencondition, can be established. Pharmacokinetic parameters of the druginfluencing the ability of a drug for treating a certain disease entityinclude, but are not limited to: half-life, volume of distribution,hepatic first-pass metabolism and the degree of blood serum binding. Theefficacy of a given drug agent can be influenced by each of theparameters mentioned above.

“Half-life” means the time where 50% of an administered drug areeliminated through biological processes, e.g. metabolism, excretion,etc. By “hepatic first-pass metabolism” is meant the propensity of adrug to be metabolized upon first contact with the liver, i.e. duringits first pass through the liver. “Volume of distribution” means thedegree of retention of a drug throughout the various compartments of thebody, like e.g. intracellular and extracellular spaces, tissues andorgans, etc. and the distribution of the drug within these compartments.“Degree of blood serum binding” means the propensity of a drug tointeract with and bind to blood serum proteins, such as albumin, leadingto a reduction or loss of biological activity of the drug.

Pharmacokinetic parameters also include bioavailability, lag time(TIag), Tmax, absorption rates, more onset and/or Cmax for a givenamount of drug administered. “Bioavailability” means the amount of adrug in the blood compartment. “Lag time” means the time delay betweenthe administration of the drug and its detection and measurability inblood or plasma. “Tmax” is the time after which maximal bloodconcentration of the drug is reached, and “Cmax” is the bloodconcentration maximally obtained with a given drug. The time to reach ablood or tissue concentration of the drug which is required for itsbiological effect is influenced by all parameters. Pharmacokineticparameters of bispecific antibody constructs exhibiting cross-speciesspecificity, which may be determined in preclinical animal testing innon-chimpanzee primates as outlined above, are also set forth e.g. inthe publication by Schlereth et al. (Cancer Immunol. Immunother. 20(2005), 1-12).

One embodiment provides the antibody construct of the invention or theantibody construct produced according to the process of the inventionfor use in the prevention, treatment or amelioration of a tumor orcancer disease or of a metastatic cancer disease.

According to a preferred embodiment of the invention said tumor orcancer disease is a solid tumor disease.

The formulations described herein are useful as pharmaceuticalcompositions in the treatment, amelioration and/or prevention of thepathological medical condition as described herein in a patient in needthereof. The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Treatment includes theapplication or administration of the formulation to the body, anisolated tissue, or cell from a patient who has a disease/disorder, asymptom of a disease/disorder, or a predisposition toward adisease/disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the disease, the symptomof the disease, or the predisposition toward the disease.

The term “amelioration” as used herein refers to any improvement of thedisease state of a patient having a tumor or cancer or a metastaticcancer as specified herein below, by the administration of an antibodyconstruct according to the invention to a subject in need thereof. Suchan improvement may also be seen as a slowing or stopping of the pprogression of the tumor or cancer or metastatic cancer of the patient.The term “prevention” as used herein means the avoidance of theoccurrence or re-occurrence of a patient having a tumor or cancer or ametastatic cancer as specified herein below, by the administration of anantibody construct according to the invention to a subject in needthereof.

The term “disease” refers to any condition that would benefit fromtreatment with the antibody construct or the pharmaceutic compositiondescribed herein. This includes chronic and acute disorders or diseasesincluding those pathological conditions that predispose the mammal tothe disease in question.

A “neoplasm” is is an abnormal growth of tissue, usually but not alwaysforming a mass. When also forming a mass, it is commonly referred to asa “tumor”. Neoplasms or tumors or can be benign, potentially malignant(pre-cancerous), or malignant. Malignant neoplasms are commonly calledcancer. They usually invade and destroy the surrounding tissue and mayform metastases, i.e., they spread to other parts, tissues or organs ofthe body. Hence, the term “metatstatic cancer” encompasses metastases toother tissues or organs than the one of the original tumor. Lymphomasand leukemias are lymphoid neoplasms. For the purposes of the presentinvention, they are also encompassed by the terms “tumor” or “cancer”.

In a preferred embodiment of the invention, the tumor or cancer diseaseis a solid tumor disease and the metastatic cancer disease can bederived from any of the foregoing.

Preferred tumor or cancer diseases in conncetion with this invention areselected from a group consisting of glioblastoma, astrocytoma,medulloblastomas, breast carcinomas, non-small cell lung carcinomas,ovarian carcinomas, prostate carcinomas, central nervous systemcarcinomas. More prefereably, the tumor or cancer disease is aglioblastoma multiforme (GBM) or an anaplastic astrocytoma. Themetastatic cancer disease can be derived from any of the foregoing.

The invention also provides a method for the treatment or ameliorationof tumor or cancer disease or a metastatic cancer disease, comprisingthe step of administering to a subject in need thereof the antibodyconstruct of the invention or the antibody construct produced accordingto the process of the invention.

The terms “subject in need” or those “in need of treatment” includesthose already with the disorder, as well as those in which the disorderis to be prevented. The subject in need or “patient” includes human andother mammalian subjects that receive either prophylactic or therapeutictreatment.

The antibody construct of the invention will generally be designed forspecific routes and methods of administration, for specific dosages andfrequencies of administration, for specific treatments of specificdiseases, with ranges of bio-availability and persistence, among otherthings. The materials of the composition are preferably formulated inconcentrations that are acceptable for the site of administration.

Formulations and compositions thus may be designed in accordance withthe invention for delivery by any suitable route of administration. Inthe context of the present invention, the routes of administrationinclude, but are not limited to

-   -   topical routes (such as epicutaneous, inhalational, nasal,        opthalmic, auricular/aural, vaginal, mucosal);    -   enteral routes (such as oral, gastrointestinal, sublingual,        sublabial, buccal, rectal); and    -   parenteral routes (such as intravenous, intraarterial,        intraosseous, intramuscular, intracerebral,        intracerebroventricular, epidural, intrathecal, subcutaneous,        intraperitoneal, extra-amniotic, intraarticular, intracardiac,        intradermal, intralesional, intrauterine, intravesical,        intravitreal, transdermal, intranasal, transmucosal,        intrasynovial, intraluminal).

The pharmaceutical compositions and the antibody construct of thisinvention are particularly useful for parenteral administration, e.g.,subcutaneous or intravenous delivery, for example by injection such asbolus injection, or by infusion such as continuous infusion.Pharmaceutical compositions may be administered using a medical device.Examples of medical devices for administering pharmaceuticalcompositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196;4,447,224; 4,447, 233; 4,486,194; 4,487,603; 4,596,556; 4,790,824;4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

In particular, the present invention provides for an uninterruptedadministration of the suitable composition. As a non-limiting example,uninterrupted or substantially uninterrupted, i.e. continuousadministration may be realized by a small pump system worn by thepatient for metering the influx of therapeutic agent into the body ofthe patient. The pharmaceutical composition comprising the antibodyconstruct of the invention can be administered by using said pumpsystems. Such pump systems are generally known in the art, and commonlyrely on periodic exchange of cartridges containing the therapeutic agentto be infused. When exchanging the cartridge in such a pump system, atemporary interruption of the otherwise uninterrupted flow oftherapeutic agent into the body of the patient may ensue. In such acase, the phase of administration prior to cartridge replacement and thephase of administration following cartridge replacement would still beconsidered within the meaning of the pharmaceutical means and methods ofthe invention together make up one “uninterrupted administration” ofsuch therapeutic agent.

The continuous or uninterrupted administration of the antibodyconstructs of the invention may be intravenous or subcutaneous by way ofa fluid delivery device or small pump system including a fluid drivingmechanism for driving fluid out of a reservoir and an actuatingmechanism for actuating the driving mechanism. Pump systems forsubcutaneous administration may include a needle or a cannula forpenetrating the skin of a patient and delivering the suitablecomposition into the patient's body. Said pump systems may be directlyfixed or attached to the skin of the patient independently of a vein,artery or blood vessel, thereby allowing a direct contact between thepump system and the skin of the patient. The pump system can be attachedto the skin of the patient for 24 hours up to several days. The pumpsystem may be of small size with a reservoir for small volumes. As anon-limiting example, the volume of the reservoir for the suitablepharmaceutical composition to be administered can be between 0.1 and 50ml.

The continuous administration may also be transdermal by way of a patchworn on the skin and replaced at intervals. One of skill in the art isaware of patch systems for drug delivery suitable for this purpose. Itis of note that transdermal administration is especially amenable touninterrupted administration, as exchange of a first exhausted patch canadvantageously be accomplished simultaneously with the placement of anew, second patch, for example on the surface of the skin immediatelyadjacent to the first exhausted patch and immediately prior to removalof the first exhausted patch. Issues of flow interruption or power cellfailure do not arise.

If the pharmaceutical composition has been lyophilized, the lyophilizedmaterial is first reconstituted in an appropriate liquid prior toadministration. The lyophilized material may be reconstituted in, e.g.,bacteriostatic water for injection (BWFI), physiological saline,phosphate buffered saline (PBS), or the same formulation the protein hadbeen in prior to lyophilization.

The compositions of the present invention can be administered to thesubject at a suitable dose which can be determined e.g. by doseescalating studies by administration of increasing doses of the antibodyconstruct of the invention exhibiting cross-species specificitydescribed herein to non-chimpanzee primates, for instance macaques. Asset forth above, the antibody construct of the invention exhibitingcross-species specificity described herein can be advantageously used inidentical form in preclinical testing in non-chimpanzee primates and asdrug in humans. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depend upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountsor doses effective for this use will depend on the condition to betreated (the indication), the delivered antibody construct, thetherapeutic context and objectives, the severity of the disease, priortherapy, the patient's clinical history and response to the therapeuticagent, the route of administration, the size (body weight, body surfaceor organ size) and/or condition (the age and general health) of thepatient, and the general state of the patient's own immune system. Theproper dose can be adjusted according to the judgment of the attendingphysician such that it can be administered to the patient once or over aseries of administrations, and in order to obtain the optimaltherapeutic effect.

A typical dosage may range from about 0.1 pg/kg to up to about 30 mg/kgor more, depending on the factors mentioned above. In specificembodiments, the dosage may range from 1.0 pg/kg up to about 20 mg/kg,optionally from 10 pg/kg up to about 10 mg/kg or from 100 pg/kg up toabout 5 mg/kg.

A therapeutic effective amount of an antibody construct of the inventionpreferably results in a decrease in severity of disease symptoms, anincrease in frequency or duration of disease symptom-free periods or aprevention of impairment or disability due to the disease affliction.For treating EGFRVIII-expressing tumors, a therapeutically effectiveamount of the antibody construct of the invention, e.g. ananti-EGFRVIII/anti-CD3 antibody construct, preferably inhibits cellgrowth or tumor growth by at least about 20%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90% relative to untreated patients. The abilityof a compound to inhibit tumor growth may be evaluated in an animalmodel predictive of efficacy in human tumors.

The pharmaceutical composition can be administered as a sole therapeuticor in combination with additional therapies such as anti-cancertherapies as needed, e.g. other proteinaceous and non-proteinaceousdrugs. These drugs may be administered simultaneously with thecomposition comprising the antibody construct of the invention asdefined herein or separately before or after administration of saidantibody construct in timely defined intervals and doses.

The term “effective and non-toxic dose” as used herein refers to atolerable dose of an inventive antibody construct which is high enoughto cause depletion of pathologic cells, tumor elimination, tumorshrinkage or stabilization of disease without or essentially withoutmajor toxic effects. Such effective and non-toxic doses may bedetermined e.g. by dose escalation studies described in the art andshould be below the dose inducing severe adverse side events (doselimiting toxicity, DLT).

The term “toxicity” as used herein refers to the toxic effects of a drugmanifested in adverse events or severe adverse events. These side eventsmight refer to a lack of tolerability of the drug in general and/or alack of local tolerance after administration. Toxicity could alsoinclude teratogenic or carcinogenic effects caused by the drug.

The term “safety”, “in vivo safety” or “tolerability” as used hereindefines the administration of a drug without inducing severe adverseevents directly after administration (local tolerance) and during alonger period of application of the drug. “Safety”, “in vivo safety” or“tolerability” can be evaluated e.g. at regular intervals during thetreatment and follow-up period. Measurements include clinicalevaluation, e.g. organ manifestations, and screening of laboratoryabnormalities. Clinical evaluation may be carried out and deviations tonormal findings recorded/coded according to NCI-CTC and/or MedDRAstandards. Organ manifestations may include criteria such asallergy/immunology, blood/bone marrow, cardiac arrhythmia, coagulationand the like, as set forth e.g. in the Common Terminology Criteria foradverse events v3.0 (CTCAE). Laboratory parameters which may be testedinclude for instance hematology, clinical chemistry, coagulation profileand urine analysis and examination of other body fluids such as serum,plasma, lymphoid or spinal fluid, liquor and the like. Safety can thusbe assessed e.g. by physical examination, imaging techniques (i.e.ultrasound, x-ray, CT scans, Magnetic Resonance Imaging (MRI), othermeasures with technical devices (i.e. electrocardiogram), vital signs,by measuring laboratory parameters and recording adverse events. Forexample, adverse events in non-chimpanzee primates in the uses andmethods according to the invention may be examined by histopathologicaland/or histochemical methods.

The above terms are also referred to e.g. in the Preclinical safetyevaluation of biotechnology-derived pharmaceuticals S6; ICH HarmonisedTripartite Guideline; ICH Steering Committee meeting on Jul. 16, 1997.

In a further embodiment, the invention provides a kit comprising anantibody construct of the invention, an antibody construct producedaccording to the process of the invention, a polynucleotide of theinvention, a vector of the invention, and/or a host cell of theinvention.

In the context of the present invention, the term “kit” means two ormore components—one of which corresponding to the antibody construct,the pharmaceutical composition, the vector or the host cell of theinvention—packaged together in a container, recipient or otherwise. Akit can hence be described as a set of products and/or utensils that aresufficient to achieve a certain goal, which can be marketed as a singleunit.

The kit may comprise one or more recipients (such as vials, ampoules,containers, syringes, bottles, bags) of any appropriate shape, size andmaterial (preferably waterproof, e.g. plastic or glass) containing theantibody construct or the pharmaceutical composition of the presentinvention in an appropriate dosage for administration (see above). Thekit may additionally contain directions for use (e.g. in the form of aleaflet or instruction manual), means for administering the antibodyconstruct of the present invention such as a syringe, pump, infuser orthe like, means for reconstituting the antibody construct of theinvention and/or means for diluting the antibody construct of theinvention.

The invention also provides kits for a single-dose administration unit.The kit of the invention may also contain a first recipient comprising adried/lyophilized antibody construct and a second recipient comprisingan aqueous formulation. In certain embodiments of this invention, kitscontaining single-chambered and multi-chambered pre-filled syringes(e.g., liquid syringes and lyosyringes) are provided.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1:

Schematic representation of EGFRvIII, which is a tumor-specific EGFRmutant found in glioblastoma with a deletion of 267 amino acids at theN-terminus.

FIG. 2:

Sequence comparison of the target binder EvIII-2 (SEQ ID NO: 169) andits derivative with covalently connected V-Regions EvIII-1 (SEQ ID NO:159).

FIGS. 3A-B:

Purification of EvIII-1 (FIG. 3B) and EvIII-2 (FIG. 3A) followingstandard research scale production

FIG. 4:

EvIII-1 and EvIII-2 monomer: Reducing SDS-PAGE

FIG. 5:

Cross-Reactivity of EvIII-1 and EvIII-2 bispecific antibody constructsshown by Flowcytometry:

Binding to human and macaque EGFRvIII and CD3

FIG. 6:

Binding of EvIII-1 and EvIII-2 bispecific antibody constructs toEGFRvIII transfected cells as well as human glioblastoma cell line U87

FIG. 7:

Cytotoxic activity of stimulated human CD8 T cells against humanEGFRvIII-transfected CHO cells. 18-hour ⁵¹chromium release assay.Effector cells: stimulated enriched human CD8 T cells. Target cells:EGFRvIII transfected CHO cells. Effector to target cell (E:T) ratio:10:1

FIG. 8:

Cytotoxic activity of stimulated human CD8 T cells against the humangliomablastoma cell line U87: (a) 18-hour ⁵¹chromium release assay.Effector cells: stimulated enriched human CD8 T cells. Target cells:vIII high positive U87 cells. Effector to target cell (E:T) ratio: 10:1;(b) 18-hour FACS based assay. Effector cells: stimulated enriched humanCD8 T cells. Target cells: hu EGFRvIII high positive U87 glioma cells.Effector to target cell (E:T) ratio: 10:1.

FIG. 9:

Cytotoxic activity of stimulated human CD8 T cells against a human tumorcell line expressing native EGFRvIII Antigen: glioblastoma cell lineDK-MG: 18-hour ⁵¹chromium release assay. Effector cells: stimulatedenriched human CD8 T cells. Target cells: DK-MG cells. Effector totarget cell (E:T) ratio: 10:1.

FIG. 10:

Cytotoxic activity of a macaque T cell line against macaqueEGFRvIII-transfected CHO cells: 48-hour FACS-based cytotoxicity assay.Effector cells: macaque CD3+LnPx4119. Target cells: macaque EGFRvIIItransfected CHO cells. Effector to target cell (E:T)-ratio: 10:1

FIG. 11:

Stability of the bispecific antibody constructs after incubation for 24Hours in human plasma:

18-hour ⁵¹Cr based assay. Effector cells: stimulated enriched human CD8T cells. Target cells: hu EGFRvIII transfected CHO cells. Effector totarget cell (E:T) ratio: 10:1. antibody constructs as indicated

FIG. 12:

The protein homogeneity of the EGFRvIII antibody constructs analyzedHigh Resolution Cation Exchange Chromatography CIEX

FIG. 13:

The surface hydrophobicity of bispecific antibody constructs tested inHydrophobic Interaction Chromatography HIC in flow-through mode

FIG. 14:

Monomer to dimer conversion after 7 days of incubation at 250 pg/ml and37° C. Analyzed with HP-SEC.

FIG. 15:

Monomer to dimer conversion after three freeze/thaw cycles at 250 pg/mland 37° C. Analyzed with HP-SEC.

FIG. 16:

FACS binding analysis of EvIII-1×CD3-scFc construct to CHO cellstransfected with the human EGFR as well as human CD3+ T cell lineHPBaLL. The red line represents cells incubated with 2 μg/ml purifiedmonomeric protein that are subsequently incubated with the mouseanti-I2C antibody and the PE labeled goat anti mouse IgG detectionantibody. The black histogram line reflects the negative control: cellsonly incubated with the anti-I2C antibody as well as the PE labeleddetection antibody

FIG. 17:

Cytotoxic activity induced by EvIII-1×CD3-scFc construct redirected toCD56 depleted unstimulated human PBMCs as effector cells and CHO cellstransfected with human EGFR as target cells. (Example 1.2)

EXAMPLES

The following examples illustrate the invention. These examples shouldnot be construed as to limit the scope of this invention. The presentinvention is limited only by the claims.

Example 1

Cytotoxic Activity

The potency of EGFRVIII×CD3 bispecific antibody constructs of theinvention in redirecting effector T cells against EGFRVIII-expressingtarget cells was analyzed in five in vitro cytotoxicity assays:

-   -   The potency of EGFRVIII×CD3 bispecific antibody constructs in        redirecting stimulated human CD8+effector T cells against human        EGFRVIII-transfected CHO cells was measured in an 18 hour ⁵¹Cr        release assay(Effector target ration 10:1). FIG. 7    -   The potency of EGFRVIII×CD3 bispecific antibody constructs in        redirecting stimulated human CD8+effector T cells against the        EGFRVIII positive human glioblastoma cell line U87 was measured        in an 18 hour ⁵¹Cr release assay(Effector target ration 10:1).        Figure    -   The potency of EGFRVII×CD3 bispecific antibody constructs in        redirecting the T cells in unstimulated human PBMC (CD14⁻/CD56⁻)        against human EGFRVIII-transfected CHO cells in the absence and        presence of soluble EGFRVIII was measured in a 48 hour        FACS-based cytotoxicity assay (Effector target ration 10:1).        FIG. 6 and Table 6    -   The potency of EGFRVII×CD3 bispecific antibody constructs in        redirecting the T cells in unstimulated human PBMC (CD14⁻/CD56⁻)        against the EGFRVIII-positive human glioblastoma cell line U87        was measured in a 48 hour FACS-based cytotoxicity assay. FIG. 8    -   For confirmation that the cross-reactive EGFRVII×CD3 bispecific        antibody constructs are capable of redirecting macaque T cells        against macaque EGFRVIII-transfected CHO cells, a 48 hour        FACS-based cytotoxicity assay was performed with a macaque T        cell line LnPx4119 as effector T cells(Effector target ration        10:1). FIG. 10

Example 1.1

Chromium Release Assay with Stimulated Human T Cells

Stimulated T cells enriched for CD8+ T cells were obtained as describedin the following. A petri dish (145 mm diameter, Greiner bio-one GmbH,Kremsmunster) was coated with a commercially available anti-CD3 specificantibody (OKT3, Orthoclone) in a final concentration of 1 pg/ml for 1hour at 37° C. Unbound protein was removed by one washing step with PBS.3-5×10⁷ human PBMC were added to the precoated petri dish in 120 ml ofRPMI 1640 with stabilized glutamine/10% FCS/IL-2 20 U/ml (Proleukin®,Chiron) and stimulated for 2 days. On the third day, the cells werecollected and washed once with RPMI 1640. IL-2 was added to a finalconcentration of 20 U/ml and the cells were cultured again for one dayin the same cell culture medium as above. CD8+cytotoxic T lymphocytes(CTLs) were enriched by depletion of CD4⁺ T cells and CD56⁺ NK cellsusing Dynal-Beads according to the manufacturer's protocol.

Cyno EGFRVIII- or human EGFRVIII-transfected CHO target cells werewashed twice with PBS and labeled with 11.1 MBq ⁵¹Cr in a final volumeof 100 μl RPMI with 50% FCS for 60 minutes at 37° C. Subsequently, thelabeled target cells were washed 3 times with 5 ml RPMI and then used inthe cytotoxicity assay. The assay was performed in a 96-well plate in atotal volume of 200 μl supplemented RPMI with an E:T ratio of 10:1. Astarting concentration of 0.01-1 pg/ml of purified bispecific antibodyconstruct and threefold dilutions thereof were used. Incubation time forthe assay was 18 hours. Cytotoxicity was determined as relative valuesof released chromium in the supernatant relative to the difference ofmaximum lysis (addition of Triton-X) and spontaneous lysis (withouteffector cells). All measurements were carried out in quadruplicates.Measurement of chromium activity in the supernatants was performed in aWizard 3″ gamma counter (Perkin Elmer Life Sciences GmbH, Koln,Germany). Analysis of the results was carried out with Prism 5 forWindows (version 5.0, GraphPad Software Inc., San Diego, Calif., USA).EC50 values calculated by the analysis program from the sigmoidal doseresponse curves were used for comparison of cytotoxic activity.

Example 1.2

Potency of Redirecting Stimulated Human Effector T Cells Against HumanEGFRVIII-Transfected CHO Cells

The cytotoxic activity of EGFRVII×CD3 bispecific antibody constructsaccording to the invention was analyzed in a 51-chromium (⁵¹Cr) releasecytotoxicity assay using CHO cells transfected with human EGFRVIII astarget cells, and stimulated human CD8+ T cells as effector cells. Theexperiment was carried out as described in Example 1.1.

The EGFRVIII×CD3 bispecific antibody constructs showed very potentcytotoxic activity against human EGFRVIII transfected CHO cells in the1-digit picomolar range.

Example 1.3

Potency of Redirecting Stimulated Human Effector T Cells Against theEGFRVIII Positive Human Cell Line Human Glioblastoma Cell Line DK-MG

The cytotoxic activity of EGFRVIII×CD3 bispecific antibody constructswas analyzed in a 51-chromium (51Cr) release cytotoxicity assay usingthe EGFRVIII-positive human glioblastoma cell line DK-MG as source oftarget cells, and stimulated human CD8+ T cells as effector cells. Theassay was carried out as described in Example 1.1.

In accordance with the results of the 51-chromium release assays withstimulated enriched human CD8+T lymphocytes as effector cells and humanEGFRVIII-transfected CHO cells as target cells, EGFRVIII×CD3 bispecificantibody constructs of the present invention are also potent incytotoxic activity against natural expresser target cells; see FIG. 9.

Example 1.4

FACS-Based Cytotoxicity Assay with Unstimulated Human PBMC

Isolation of Effector Cells

Human peripheral blood mononuclear cells (PBMC) were prepared by Ficolldensity gradient centrifugation from enriched lymphocyte preparations(buffy coats), a side product of blood banks collecting blood fortransfusions. Buffy coats were supplied by a local blood bank and PBMCwere prepared on the same day of blood collection. After Ficoll densitycentrifugation and extensive washes with Dulbecco's PBS (Gibco),remaining erythrocytes were removed from PBMC via incubation witherythrocyte lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 100 pM EDTA).Platelets were removed via the supernatant upon centrifugation of PBMCat 100×g. Remaining lymphocytes mainly encompass B and T lymphocytes, NKcells and monocytes. PBMC were kept in culture at 37° C./5% CO₂ in RPMImedium (Gibco) with 10% FCS (Gibco).

Depletion of CD14⁺ and CD56⁺ Cells

For depletion of CD14⁺ cells, human CD14 MicroBeads (Milteny Biotec,MACS, #130-050-201) were used, for depletion of NK cells human CD56MicroBeads (MACS, #130-050-401). PBMC were counted and centrifuged for10 min at room temperature with 300×g. The supernatant was discarded andthe cell pellet resuspended in MACS isolation buffer [80 μL/10⁷ cells;PBS (Invitrogen, #20012-043), 0.5% (v/v) FBS (Gibco, #10270-106), 2 mMEDTA (Sigma-Aldrich, # E-6511)]. CD14 MicroBeads and CD56 MicroBeads (20μL/10⁷ cells) were added and incubated for 15 min at 4-8° C. The cellswere washed with MACS isolation buffer (1-2 mL/10⁷ cells). Aftercentrifugation (see above), supernatant was discarded and cellsresuspended in MACS isolation buffer (500 μL/10⁸ cells). CD14/CD56negative cells were then isolated using LS Columns (Miltenyi Biotec,#130-042-401). PBMC w/o CD14+/CD56+cells were cultured in RPMI completemedium i.e. RPMI1640 (Biochrom AG, # FG1215) supplemented with 10% FBS(Biochrom AG, # S0115), 1× non-essential amino acids (Biochrom AG, #K0293), 10 mM Hepes buffer (Biochrom AG, # L1613), 1 mM sodium pyruvate(Biochrom AG, # L0473) and 100 U/mL penicillin/streptomycin (BiochromAG, # A2213) at 37° C. in an incubator until needed.

Target Cell Labeling

For the analysis of cell lysis in flow cytometry assays, the fluorescentmembrane dye DiOC₁₈ (DiO) (Molecular Probes, # V22886) was used to labelhuman EGFRVIII- or macaque EGFRVIII-transfected CHO cells as targetcells and distinguish them from effector cells. Briefly, cells wereharvested, washed once with PBS and adjusted to 10⁶ cell/mL in PBScontaining 2% (v/v) FBS and the membrane dye DiO (5 μL/10⁶ cells). Afterincubation for 3 min at 37° C., cells were washed twice in complete RPMImedium and the cell number adjusted to 1.25×10⁵ cells/mL. The vitalityof cells was determined using 0.5% (v/v) isotonic EosinG solution (Roth,#45380).

Flow Cytometry Based Analysis

This assay was designed to quantify the lysis of cyno or humanEGFRVIII-transfected CHO cells in the presence of serial dilutions ofEGFRVIII bispecific antibody constructs. Equal volumes of DiO-labeledtarget cells and effector cells (i.e., PBMC w/o CD14⁺ cells) were mixed,resulting in an E:T cell ratio of 10:1. 160 μl of this suspension weretransferred to each well of a 96-well plate. 40 μL of serial dilutionsof the EGFRVII×CD3 bispecific antibody constructs and a negative controlbispecific (a CD3-based bispecific antibody construct recognizing anirrelevant target antigen) or RPMI complete medium as an additionalnegative control were added. The bispecific antibody-mediated cytotoxicreaction proceeded for 48 hours in a 7% CO₂ humidified incubator. Thencells were transferred to a new 96-well plate and loss of target cellmembrane integrity was monitored by adding propidium iodide (PI) at afinal concentration of 1 pg/mL. PI is a membrane impermeable dye thatnormally is excluded from viable cells, whereas dead cells take it upand become identifiable by fluorescent emission.

Samples were measured by flow cytometry on a FACSCanto II instrument andanalyzed by FACSDiva software (both from Becton Dickinson). Target cellswere identified as DiO-positive cells. PI-negative target cells wereclassified as living target cells. Percentage of cytotoxicity wascalculated according to the following formula:

${{Cytotoxicity}\mspace{14mu}\lbrack\%\rbrack} = {\frac{n_{{dead}\mspace{14mu} {target}\mspace{14mu} {cells}}}{n_{{target}\mspace{14mu} {cells}}} \times 100}$n = number  of  events

Using GraphPad Prism 5 software (Graph Pad Software, San Diego), thepercentage of cytotoxicity was plotted against the correspondingbispecific antibody construct concentrations. Dose response curves wereanalyzed with the four parametric logistic regression models forevaluation of sigmoid dose response curves with fixed hill slope andEC50 values were calculated.

Example 1.5

Potency of Redirecting Unstimulated Human PBMC Against HumanEGFRVIII-Transfected CHO Cells in Absence and Presence of SolubleEGFRVIII

The cytotoxic activity of EGFRVIII×CD3 bispecific antibody constructswas analyzed in a FACS-based cytotoxicity assay using CHO cellstransfected with human EGFRVIII as target cells, and unstimulated humanPBMC as effector cells. The assay was carried out as described inExample 1.4 above.

Expectedly, EC50 values were generally higher in cytotoxicity assayswith unstimulated PBMC as effector cells compared with cytotoxicityassays using stimulated human CD8+ T cells (see Example 1.2).

Example 1.6

Potency of Redirecting Unstimulated Human PBMC Against theEGFRVIII-Positive Human Glioblastoma Cell Line U87 or DK-MG Cells

The cytotoxic activity of EGFRVIII×CD3 bispecific antibody constructswas furthermore analyzed in a FACS-based cytotoxicity assay using theEGFRVIII-positive human glioblastoma cell line U87 or DK-MG as a sourceof target cells and unstimulated human PBMC as effector cells. The assaywas carried out as described in Example 1.4 above. The results are shownin FIGS. 8 and 9.

Example 1.7

Potency of Redirecting Macaque T Cells Against MacaqueEGFRVIII-Expressing CHO Cells

The cytotoxic activity of EGFRVIII×CD3 bispecific antibody constructswas analyzed in a FACS-based cytotoxicity assay using CHO cellstransfected with macaque (cyno) EGFRVIII as target cells, and themacaque T cell line 4119LnPx (Knappe et al. Blood 95:3256-61 (2000)) assource of effector cells. Target cell labeling of macaqueEGFRVIII-transfected CHO cells and ⁵¹Cr-relase based analysis ofcytotoxic activity was performed as described above.

Results are shown in FIG. 10. Macaque T cells from cell line 4119LnPxwere induced to efficiently kill macaque EGFRVIII-transfected CHO cellsby EGFRVIII×CD3 bispecific antibody constructs of the invention.

Example 1.8

Potency Gap Between the Monomeric and the Dimeric Isoform of BispecificAntibody Constructs

In order to determine the difference in cytotoxic activity between themonomeric and the dimeric isoform of individual EGFRVIII×CD3 bispecificantibody constructs (referred to as potency gap), an 18 hour 51-chromiumrelease cytotoxicity assay was carried out as described hereinabove(Example 1.1) with purified bispecific antibody construct monomer anddimer. Effector cells were stimulated enriched human CD8+ T cells.Target cells were hu EGFRVIII transfected CHO cells. Effector to targetcell (E:T) ratio was 10:1. The potency gap was calculated as ratiobetween EC50 values.

Example 2

Stability after Incubation for 24 Hours in Human Plasma

Purified bispecific antibody constructs were incubated at a ratio of 1:5in a human plasma pool at 37° C. for 96 hours at a final concentrationof 2-20 pg/ml. After plasma incubation the antibody constructs werecompared in a 51-chromium release assay with stimulated enriched humanCD8+ T cells and human EGFRVIII-transfected CHO cells at a startingconcentration of 0.01-0.1 pg/ml and with an effector to target cell(E:T) ratio of 10:1 (assay as described in Example 1.1). Non-incubated,freshly thawed bispecific antibody constructs were included as controls.

The results are shown in FIG. 11; The antibody construct EvIII-2 had aplasma stability (EC₅₀ plasma/EC₅₀ control) of around 2. Surprisingly,the bispecific antibody construct of the invention did show almost noconversion.

Example 3

Protein Homogeneity by High Resolution Cation Exchange Chromatography

The protein homogeneity the antibody constructs of the invention wasanalyzed by high resolution cation exchange chromatography CIEX.

50 μg of antibody construct monomer were diluted with 50 ml bindingbuffer A (20 mM sodium dihydrogen phosphate, 30 mM NaCl, 0.01% sodiumoctanate, pH 5.5), and 40 ml of this solution were applied to a 1 mlBioPro SP-F column (YMC, Germany) connected to an Äkta Micro FPLC device(GE Healthcare, Germany). After sample binding, a wash step with furtherbinding buffer was carried out. For protein elution, a linear increasingsalt gradient using buffer B (20 mM sodium dihydrogen phosphate, 1000 mMNaCl, 0.01% sodium octanate, pH 5.5) up to 50% percent buffer B wasapplied over 10 column volumes. The whole run was monitored at 280, 254and 210 nm optical absorbance. Analysis was done by peak integration ofthe 280 nm signal recorded in the Äkta Unicorn software run evaluationsheet.

The results are shown in FIG. 12. Almost all tested antibody constructshave a very favourable homogeneity of ≥95% (area under the curve (=AUC)of the main peak)..

Example 4

Surface Hydrophobicity as Measured by HIC Butyl

The surface hydrophobicity of bispecific antibody constructs of theinvention was tested in Hydrophobic Interaction Chromatography HIC inflow-through mode.

50 μg of antibody construct monomer were diluted with genericformulation buffer to a final volume of 500 μl (10 mM citric acid, 75 mMlysine HCl, 4% trehalose, pH 7.0) and applied to a 1 ml Butyl SepharoseFF column (GE Healthcare, Germany) connected to a Äkta Purifier FPLCsystem (GE Healthcare, Germany). The whole run was monitored at 280, 254and 210 nm optical absorbance. Analysis was done by peak integration ofthe 280 nm signal recorded in the Äkta Unicorn software run evaluationsheet. Elution behavior was evaluated by comparing area and velocity ofrise and decline of protein signal thereby indicating the strength ofinteraction of the BiTE albumin fusion with the matrix.

The antibody constructs had a good elution behaviour, which was mostlyrapid and complete; see FIG. 13.

Example 5

Monomer to Dimer Conversion after (i) Three Freeze/Thaw Cycles and (ii)7 Days of Incubation at 250 μg/Ml

Bispecific EGFRVIII×CD3 antibody monomeric construct were subjected todifferent stress conditions followed by high performance SEC todetermine the percentage of initially monomeric antibody construct,which had been converted into dimeric antibody construct.

-   (i) 25 μg of monomeric antibody construct were adjusted to a    concentration of 250 μg/ml with generic formulation buffer and then    frozen at −80° C. for 30 min followed by thawing for 30 min at room    temperature. After three freeze/thaw cycles the dimer content was    determined by HP-SEC.-   (ii) 25 μg of monomeric antibody construct were adjusted to a    concentration of 250 μg/ml with generic formulation buffer followed    by incubation at 37° C. for 7 days. The dimer content was determined    by HP-SEC.

A high resolution SEC Column TSK Gel G3000 SWXL (Tosoh, Tokyo-Japan) wasconnected to an Äkta Purifier 10 FPLC (GE Lifesciences) equipped with anA905 Autosampler. Column equilibration and running buffer consisted of100 mM KH2PO4-200 mM Na2SO4 adjusted to pH 6.6. The antibody solution(25 μg protein) was applied to the equilibrated column and elution wascarried out at a flow rate of 0.75 ml/min at a maximum pressure of 7MPa. The whole run was monitored at 280, 254 and 210 nm opticalabsorbance. Analysis was done by peak integration of the 210 nm signalrecorded in the Äkta Unicorn software run evaluation sheet. Dimercontent was calculated by dividing the area of the dimer peak by thetotal area of monomer plus dimer peak.

The results are shown in FIGS. 14 and 15 below. The EVIII-1×CD3bispecific antibody constructs of the invention presented with dimerpercentages of 0.59% after three freeze/thaw cycles (FIG. 15), and withdimer percentages of 0.26% after 7 days of incubation at 37° C. whilethe EvIII-1×CD3 bispecific antibody construct showed higher values of1.56% after 7 days and 2.53% after three freeze/thaw cycles.

Example 6

Thermostability

Antibody aggregation temperature was determined as follows: 40 μl ofantibody construct solution at 250 μg/ml were transferred into a singleuse cuvette and placed in a Wyatt Dynamic Light Scattering deviceDynaPro Nanostar (Wyatt). The sample was heated from 40° C. to 70° C. ata heating rate of 0.5° C./min with constant acquisition of the measuredradius. Increase of radius indicating melting of the protein andaggregation was used by the software package delivered with the DLSdevice to calculate the aggregation temperature of the antibodyconstruct.

TABLE 2 Thermostability of the bispecific antibody constructs asdetermined by DLS (dynamic light scattering) EGFRVIII Thermal StabilityHALB BiTE DLS T_(A) [° C.] EvIII-1 51.6 EvIII-2 51.2

Example 7

Turbidity at 2500 μg/Ml Antibody Concentration

1 ml of purified antibody construct solution of a concentration of 250μg/ml was concentrated by spin concentration units to 2500 μg/ml. After16h storage at 5° C. the turbidity of the antibody solution wasdetermined by OD340 nm optical absorption measurement against thegeneric formulation buffer.

The results are shown in Table 3 below. While the EvIII-1 antibodyconstruct of the invention had an extreme favourable turbidity of ≤0.03the EvIII-2 antibody construct showed significant turbidity, which isindicative for less favourable characteristics in the formulation ofsuch molecule in a pharmaceutical composition.

TABLE 3 Turbidity of the antibody constructs after concentration to 2.5mg/ml over night EGFRVIII Turbidity after 16 h @ HALB BiTE 2500 μg/ml[OD340] EvIII-1 0.029 EvIII-2 2.87

TABLE 4 Sequence Listing SEQ ID NO: Description Source Sequence 1.Peptide artificial GGGG linker 2. Peptide artificial GGGGS linker 3.Peptide artificial GGGGQ linker 4. Peptide artificial PGGGGS linker 5.Peptide artificial PGGDGS linker 6. Peptide artificial SGGGGS linker 7.Peptide artificial GGGGS GGGS linker 8. Peptide artificial GGGGS GGGGSlinker 9. Peptide artificial GGGGS GGGGS GGGGS linker 10. Hexa-artificial HHHHHH histidine 11. CDR-L1 of artificial GSSTGAVTSGYYPN F6A12. CDR-L2 of artificial GTKFLAP F6A 13. CDR-L3 of artificial ALWYSNRWVF6A 14. CDR-H1 of artificial IYAMN F6A 15. CDR-H2 of artificialRIRSKYNNYATYYADSVKS F6A 16. CDR-H3 of artificial HGNFGNSYVSFFAY F6A 17.VH of F6A artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSS 18. VL of F6AartificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 19. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK F6ASRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 20. CDR-L1 ofartificial GSSTGAVTSGYYPN H2C 21. CDR-L2 of artificial GTKFLAP H2C 22.CDR-L3 of artificial ALWYSNRWV H2C 23. CDR-H1 of artificial KYAMN H2C24. CDR-H2 of artificial RIRSKYNNYATYYADSVKD H2C 25. CDR-H3 ofartificial HGNFGNSYISYWAY H2C 26. VH of H2C artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 27. VL of H2CartificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSQLLGGKAALTLSGVPEDEAEYYCALWYSNRWVFGGGTKLTVL 28. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK H2CDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 29. CDR-L1 ofartificial GSSTGAVTSGYYPN H1E 30. CDR-L2 of artificial GTKFLAP H1E 31.CDR-L3 of artificial ALWYSNRWV H1E 32. CDR-H1 of artificial SYAMN H1E33. CDR-H2 of artificial RIRSKYNNYATYYADSVKG H1E 34. CDR-H3 ofartificial HGNFGNSYLSFWAY H1E 35. VH of H1E artificialEVQLVESGGGLEQPGGSLKLSCAASGETENSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSS 36. VL of H1EartificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 37. VH-VL of artificialEVQLVESGGGLEQPGGSLKLSCAASGETENSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK H1EGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 38. CDR-L1 ofartificial GSSTGAVTSGYYPN G4H 39. CDR-L2 of artificial GTKFLAP G4H 40.CDR-L3 of artificial ALWYSNRWV G4H 41. CDR-H1 of artificial RYAMN G4H42. CDR-H2 of artificial RIRSKYNNYATYYADSVKG G4H 43. CDR-H3 ofartificial HGNFGNSYLSYFAY G4H 44. VH of G4H artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSS 45. VL of G4HartificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 46. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK G4HGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 47. CDR-L1 ofartificial RSSTGAVTSGYYPN A2J 48. CDR-L2 of artificial ATDMRPS A2J 49.CDR-L3 of artificial ALWYSNRWV A2J 50. CDR-H1 of artificial VYAMN A2J51. CDR-H2 of artificial RIRSKYNNYATYYADSVKK A2J 52. CDR-H3 ofartificial HGNFGNSYLSWWAY A2J 53. VH of A2J artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSS 54. VL of A2JartificialQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 55. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK A2JKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 56. CDR-L1 ofartificial GSSTGAVTSGYYPN E1L 57. CDR-L2 of artificial GTKFLAP E1L 58.CDR-L3 of artificial ALWYSNRWV E1L 59. CDR-H1 of artificial KYAMN E1L60. CDR-H2 of artificial RIRSKYNNYATYYADSVKS E1L 61. CDR-H3 ofartificial HGNFGNSYTSYYAY E1L 62. VH of E1L artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSS 63. VL of E1LartificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 64. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK E1LSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 65. CDR-L1 ofartificial RSSTGAVTSGYYPN E2M 66. CDR-L2 of artificial ATDMRPS E2M 67.CDR-L3 of artificial ALWYSNRWV E2M 68. CDR-H1 of artificial GYAMN E2M69. CDR-H2 of artificial RIRSKYNNYATYYADSVKE E2M 70. CDR-H3 ofartificial HRNFGNSYLSWFAY E2M 71. VH of E2M artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSS 72. VL of E2MartificialQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 73. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK E2MERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARESGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 74. CDR-L1 ofartificial GSSTGAVTSGYYPN F70 75. CDR-L2 of artificial GTKFLAP F70 76.CDR-L3 of artificial ALWYSNRWV F70 77. CDR-H1 of artificial VYAMN F7078. CDR-H2 of artificial RIRSKYNNYATYYADSVKK F70 79. CDR-H3 ofartificial HGNEGNSYISWWAY F70 80. VH of F70 artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSS 81. VL of F70artificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 82. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK F70KRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 83. CDR-L1 ofartificial GSSTGAVTSGNYPN F12Q 84. CDR-L2 of artificial GTKFLAP F12Q 85.CDR-L3 of artificial VLWYSNRWV F12Q 86. CDR-H1 of artificial SYAMN F12Q87. CDR-H2 of artificial RIRSKYNNYATYYADSVKG F12Q 88. CDR-H3 ofartificial HGNFGNSYVSWWAY F12Q 89. VH of F12Q artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS 90.VL of F12Q artificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 91. VH-VL of artificialEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK F12QGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 92. CDR-L1 ofartificial GSSTGAVTSGNYPN I2C 93. CDR-L2 of artificial GTKFLAP I2C 94.CDR-L3 of artificial VLWYSNRWV I2C 95. CDR-H1 of artificial KYAMN I2C96. CDR-H2 of artificial RIRSKYNNYATYYADSVKD I2C 97. CDR-H3 ofartificial HGNEGNSYISYWAY I2C 98. VH of I2C artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSYISYWAYWGQGTLVTVSS 99. VL of I2CartificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 100. VH-VL of  artificialEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK I2CDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 101. VH of F12qartificialEVQLVESGGGLVQPGGSLRLSCAASGETENSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS 102.VL of F12q artificialQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 103. F12q scFvEVQLVESGGGLVQPGGSLRLSCAASGETENSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 104. HALB humanDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 105. HALB7 artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 106. HALB098artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 107. HALB114artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLVAASKAALGL 108. HALB254artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 109. HALB253artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLYEIARRHPYFYAPELLFFAKRLKKYYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 110. HALB131artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPHLVAASQAALGL 111. HALB135artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLVAASKAALGL 112. HALB133artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASKAALGL 113. HALB234artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASKAALGL 114. HALB C34SartificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 115. HALB7 C34SartificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 116. HALB098artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 117. HALB114artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLVAASKAALGL 118. HALB254artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 119. HALB253artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 120. HALB131artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPHLVAASQAALGL 121. HALB135artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLVAASKAALGL 122. HALB133artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASKAALGL 123. HALB234artificialDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34STLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASKAALGL 124. HALB C34AartificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 125. HALB7 C34AartificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 126. HALB098artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 127. HALB114artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLVAASKAALGL 128. HALB254artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 129. HALB253artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL 130. HALB131artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPHLVAASQAALGL 131. HALB135artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLVAASKAALGL 132. HALB133artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLVAASKAALGL 133. HALB234artificialDAHKSEVAHREKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLH C34ATLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASKAALGL 134. Ab156 artificialRDWDFDVFGGGTPVGG 135. linear FcRn artificial QRFVTGHFGGLXPANG bindingpeptide 136. linear FcRn artificial QRFVTGHFGGLYPANG binding peptide Y137. linear FcRn artificial QRFVTGHFGGLHPANG binding peptide H 138.core FcRn artificial TGHFGGLHP binding peptide H 139. cyclic FcRnartificial QRFCTGHFGGLHPCNG binding peptide H 140. Cross body 1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS HCVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 141.Cross body 1GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA LCASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 142.Cross body 2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS HCVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 143.Cross body 2GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA LCASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK144. Hetero-FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAbinder FcKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 145. Hetero-FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNApartner FcKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 146. Maxibody 1EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVtarget FcEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQ 147. Maxibody 1YGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQCD3 FcVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 148. Maxibody 2EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVtarget FcEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 149.  Maxibody2EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVCD3 FcEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 150. Mono FcAPELLGGPSVFLFPPKPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK 151. EvIII-1 VH CDR1 NYGMH 152. EvIII-1 VH CDR2VIWYDGSDKYYADSVRG 153. EvIII-1 VH CDR3 DGYDILTGNPRDFDY 154. EvIII-1VL CDR1 RSSQSLVHSDGNTYLS 155. EvIII-1 VL CDR2 RISRRFS 156. EvIII-1VL CDR3 MQSTHVPRT 157. EvIII-1 VHQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSS 158. EvIII-1VL DTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGCGTKVEIK 159. EvIII-1 scFvQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGCGTKVEIK 160. EvIII-1bispecificQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRmoleculeFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 161. EvIII-2 VH CDR1 NYGMH 162.EvIII-2 VH CDR2 VIWYDGSDKYYADSVRG 163. EvIII-2 VH CDR3 DGYDILTGNPRDFDY164. EvIII-2 VL CDR1 RSSQSLVHSDGNTYLS 165. EvIII-2 VL CDR2 RISRRFS 166.EvIII-2 VL CDR3 MQSTHVPRT 167. EvIII-2 VHQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKGLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSS 168. EvIII-2VL DTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGQGTKVEIK 169. EvIII-2 scFvQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKGLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGQGTKVEIK 170. EvIII-2bispecificQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKGLEWVAVIWYDGSDKYYADSVRGRmoleculeFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGQGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 171. EGFR vIII humanLEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA 172. EGFR vIIIcynomolgusLEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDTLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSSQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCQNVSRGRECVDKCNILEGEPREFVENSECIQCHPECLPQVMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCARNGPKIPSIATGMLGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA 173. Fc monomer-1artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA+c/−gKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 174. Fc monomer-2 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA+c/−g/delGKKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 175. Fc monomer-3 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA−c/+gKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKLSLSPGK 176. Fc monomer-4 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA−c/+g/delGKKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 177. Fc monomer-5 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA−c/−gKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 178. Fc monomer-6 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA−c/−g/delGKKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 179. Fc monomer-7 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA+c/+gKTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 180. Fc monomer-8 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA+c/+g/delGKKTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 181. scFc-1 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 182. scFc-2 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 183. scFc-3 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 184. scFc-4 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 185. scFc-5 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 186. scFc-6 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 187. scFc-7 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 188. scFc-8 artificialDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 189. EvIII-1xCD3- BispecificQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGR scFcHLE FTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSmoleculeGGGGSDTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 190.EvIII-1xCD3- BispecificQVQLVESGGGVVQSGRSLRLSCAASGETFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRscFc_delGK HLEFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSmoleculeGGGGSDTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPRTFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGETFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARESGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 191. Peptide(G₄S)₄ GGGGSGGGGSGGGGSGGGGS linker linker 192. Peptide (G₄S)₅GGGGSGGGGSGGGGSGGGGSGGGGS linker linker 193. Peptide (G₄S)₆GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker linker 194. Peptide (G₄S)₇GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker linker 195. Peptide (G₄S)₈GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker linker

1-14. (canceled)
 15. A method for treating glioblastoma or glioma,comprising administering to a subject in need thereof an effectiveamount of a bispecific antibody construct, said antibody constructcomprises a first binding domain which binds to human and macaqueepidermal growth factor receptor VIII (EGFRVIII) on the surface of atarget cell and a second binding domain which binds to human CD3 on thesurface of a T cell, wherein the first binding domain comprises a heavychain variable region (VH) comprising the amino acid sequence of SEQ IDNO: 157, and a light chain variable region (VL) comprising the aminoacid sequence of SEQ ID NO:
 158. 16. The method of claim 15, whereinsaid antibody construct is in a format selected from the groupconsisting of (scFv)₂, diabodies, and oligomers thereof.
 17. The methodof claim 15, wherein said second binding domain binds to human CD3epsilon and to Callithrix jacchus, Saguinus oedipus or Saimiri sciureusCD3 epsilon, and comprises: (i) a VL region comprising: a CDR-L1comprising the amino acid of SEQ ID NO: 11, a CDR-L2 comprising theamino acid of SEQ ID NO:12, and a CDR-L3 comprising the amino acid ofSEQ ID NO:13; and a VH region comprising: a CDR-H1 comprising the aminoacid of SEQ ID NO: 14, a CDR-H2 comprising the amino acid of SEQ IDNO:15, and a CDR-H3 comprising the amino acid of SEQ ID NO:16; (ii) a VLregion comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 20,a CDR-L2 comprising the amino acid of SEQ ID NO:21, and a CDR-L3comprising the amino acid of SEQ ID NO:22; and a VH region comprising: aCDR-H1 comprising the amino acid of SEQ ID NO: 23, a CDR-H2 comprisingthe amino acid of SEQ ID NO:24, and a CDR-H3 comprising the amino acidof SEQ ID NO:25; (iii) a VL region comprising: a CDR-L1 comprising theamino acid of SEQ ID NO: 29, a CDR-L2 comprising the amino acid of SEQID NO:30, and a CDR-L3 comprising the amino acid of SEQ ID NO:31; and aVH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO:32, a CDR-H2 comprising the amino acid of SEQ ID NO:33, and a CDR-H3comprising the amino acid of SEQ ID NO:34; (iv) a VL region comprising:a CDR-L1 comprising the amino acid of SEQ ID NO: 38, a CDR-L2 comprisingthe amino acid of SEQ ID NO:39, and a CDR-L3 comprising the amino acidof SEQ ID NO:40; and a VH region comprising: a CDR-H1 comprising theamino acid of SEQ ID NO: 41, a CDR-H2 comprising the amino acid of SEQID NO:42, and a CDR-H3 comprising the amino acid of SEQ ID NO:43; (v) aVL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO:47, a CDR-L2 comprising the amino acid of SEQ ID NO:48, and a CDR-L3comprising the amino acid of SEQ ID NO:49; and a VH region comprising: aCDR-H1 comprising the amino acid of SEQ ID NO: 50, a CDR-H2 comprisingthe amino acid of SEQ ID NO:51, and a CDR-H3 comprising the amino acidof SEQ ID NO:52; (vi) a VL region comprising: a CDR-L1 comprising theamino acid of SEQ ID NO: 56, a CDR-L2 comprising the amino acid of SEQID NO:57, and a CDR-L3 comprising the amino acid of SEQ ID NO:58; and aVH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO:59, a CDR-H2 comprising the amino acid of SEQ ID NO:60, and a CDR-H3comprising the amino acid of SEQ ID NO:61; (vii) a VL region comprising:a CDR-L1 comprising the amino acid of SEQ ID NO: 65, a CDR-L2 comprisingthe amino acid of SEQ ID NO:66, and a CDR-L3 comprising the amino acidof SEQ ID NO:67; and a VH region comprising: a CDR-H1 comprising theamino acid of SEQ ID NO: 68, a CDR-H2 comprising the amino acid of SEQID NO:69, and a CDR-H3 comprising the amino acid of SEQ ID NO:70; (viii)a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO:74, a CDR-L2 comprising the amino acid of SEQ ID NO:75, and a CDR-L3comprising the amino acid of SEQ ID NO:76; and a VH region comprising: aCDR-H1 comprising the amino acid of SEQ ID NO: 77, a CDR-H2 comprisingthe amino acid of SEQ ID NO:78, and a CDR-H3 comprising the amino acidof SEQ ID NO:79; (ix) a VL region comprising: a CDR-L1 comprising theamino acid of SEQ ID NO: 83, a CDR-L2 comprising the amino acid of SEQID NO:84, and a CDR-L3 comprising the amino acid of SEQ ID NO:85; and aVH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO:86, a CDR-H2 comprising the amino acid of SEQ ID NO:87, and a CDR-H3comprising the amino acid of SEQ ID NO:88; (x) a VL region comprising: aCDR-L1 comprising the amino acid of SEQ ID NO: 92, a CDR-L2 comprisingthe amino acid of SEQ ID NO:93, and a CDR-L3 comprising the amino acidof SEQ ID NO:94; and a VH region comprising: a CDR-H1 comprising theamino acid of SEQ ID NO: 95, a CDR-H2 comprising the amino acid of SEQID NO:96, and a CDR-H3 comprising the amino acid of SEQ ID NO:97; or(xi) a VH region comprising amino acid sequence of SEQ ID NO: 101, and aVL region comprising amino acid sequence of SEQ ID NO:
 102. 18. Themethod of claim 15, wherein said antibody construct comprises: (a) apolypeptide comprising in the following order starting from theN-terminus: (i) a polypeptide comprising the amino acid sequence of SEQID NO: 159; (ii) a peptide linker comprising the amino acid sequenceselected from any one of SEQ ID NOs: 1-9; (iii) a polypeptide comprisingthe amino acid sequence selected from the group consisting of: SEQ IDNO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100,and SEQ ID NO: 103; and (iv) optionally, a His-tag comprising the aminoacid sequence of SEQ ID NO: 10; (b) a polypeptide comprising infollowing order starting from the N-terminus: (i) a polypeptidecomprising the amino acid sequence of SEQ ID NO: 159; (ii) a peptidelinker comprising the amino acid sequence selected from any one of SEQID NOs: 1-9; (iii) a polypeptide comprising the amino acid sequenceselected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73,SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; (iv)optionally, a peptide linker comprising the amino acid sequence selectedfrom any one of SEQ ID NOs: 1-9; (v) a polypeptide comprising the aminoacid sequence of SEQ ID NO:134; and (vi) optionally, a His-tagcomprising the amino acid sequence of SEQ ID NO: 10; (c) a polypeptidecomprising in the following order starting from the N-terminus: (i) apolypeptide comprising the amino acid sequence QRFVTGHFGGLX₁PANG (SEQ IDNO: 135) whereas X₁ is Y or H; (ii) a polypeptide comprising the aminoacid sequence of: SEQ ID NO: 159; (iii) a peptide linker comprising theamino acid sequence selected from any one of SEQ ID NOs: 1-9; (iv) apolypeptide comprising the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO:46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ IDNO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; (v) a polypeptide comprisingthe amino acid sequence QRFVTGHFGGLHPANG (SEQ ID NO: 137) orQRFCTGHFGGLHPCNG (SEQ ID NO: 139); and (vi) optionally, a His-tagcomprising the amino acid sequence of SEQ ID NO: 10; (d) a firstpolypeptide comprising in the following order starting from theN-terminus: (i) a polypeptide comprising the amino acid sequenceselected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 26, SEQID NO: 35, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71,SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98, and SEQ ID NO: 101; (ii) apeptide linker comprising the amino acid sequence of SEQ ID NO: 8; (iii)a polypeptide comprising the amino acid of SEQ ID NO: 158; and (iv) apolypeptide comprising the amino acid sequence of SEQ ID NO: 140; and asecond polypeptide comprising in the following order starting from theN-terminus: (i) a polypeptide comprising the amino acid sequence of SEQID NO: 157; (ii) a peptide linker comprising the amino acid sequence ofSEQ ID NO: 8; (iii) a polypeptide comprising the amino acid sequenceselected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 27, SEQID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72,SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, and SEQ ID NO: 102, and aserine residue at the C-terminus; and (iv) a polypeptide comprising theamino acid sequence of SEQ ID NO: 141; (e) a first polypeptidecomprising in the following order starting from the N-terminus: (i) apolypeptide comprising the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO:44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 80, SEQ IDNO: 89, SEQ ID NO: 98, and SEQ ID NO: 101; (ii) a peptide linkercomprising the amino acid sequence of SEQ ID NO: 8; (iii) a polypeptidecomprising the amino acid sequence of SEQ ID NO: 158; and (iv) apolypeptide comprising the amino acid sequence of SEQ ID NO: 142; and asecond polypeptide comprising in the following order starting from theN-terminus: (i) a polypeptide comprising the amino acid sequence of SEQID NO: 157; (ii) a peptide linker comprising the amino acid sequence ofSEQ ID NO: 8; (iii) a polypeptide comprising the amino acid sequenceselected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 27, SEQID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72,SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, and SEQ ID NO: 102, and aserine residue at the C-terminus; and (iv) a polypeptide comprising theamino acid sequence of SEQ ID NO: 143; (f) a first polypeptidecomprising in the following order starting from the N-terminus: (i) apolypeptide comprising the amino acid sequence of SEQ ID NO: 159; (ii) apeptide linker comprising the amino acid sequence selected from any oneof SEQ ID NOs: 1-9; (iii) a polypeptide comprising the amino acidsequence selected from the group consisting of: SEQ ID NO: 19, SEQ IDNO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO:103; and (iv) a polypeptide comprising the amino acid sequence of SEQ IDNO: 144; and a second polypeptide comprising the amino acid sequence ofSEQ ID NO: 145; (g) a first polypeptide comprising in the followingorder starting from the N-terminus: (i) a polypeptide comprising theamino acid sequence of SEQ ID NO: 159; and (ii) a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 146; and a second polypeptidecomprising in the following order starting from the N-terminus: (i) apolypeptide comprising the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO:46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ IDNO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and (ii) a polypeptidecomprising the amino acid sequence of SEQ ID NO: 147; (h) a firstpolypeptide comprising in the following order starting from theN-terminus: (i) a polypeptide comprising the amino acid sequence of SEQID NO: 159; and (ii) a polypeptide comprising the amino acid sequence ofSEQ ID NO: 148; and a second polypeptide comprising in the followingorder starting from the N-terminus: (i) a polypeptide comprising theamino acid sequence selected from the group consisting of: SEQ ID NO:19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ IDNO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, andSEQ ID NO: 103; and (ii) a polypeptide comprising the amino acidsequence of SEQ ID NO: 149; (i) a polypeptide comprising in thefollowing order starting from the N-terminus: (i) a polypeptidecomprising the amino acid sequence of SEQ ID NO: 159; (ii) a peptidelinker comprising the amino acid sequence selected from any one of SEQID NOs: 1-9; (iii) a polypeptide comprising the amino acid sequenceselected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73,SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and(iv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 150;or (j) a polypeptide comprising in the following order starting from theN-terminus: (i) a polypeptide comprising the amino acid sequence of SEQID NO: 159; (ii) a peptide linker comprising the amino acid sequenceselected from any one of SEQ ID NOs: 1-9; (iii) a polypeptide comprisingthe amino acid sequence selected from the group consisting of: SEQ IDNO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100,and SEQ ID NO: 103; (iv) a peptide linker having an amino acid sequenceselected from the group consisting of: SEQ ID NOs: 1, 2, 4, 5, 6, 8 and9; and (v) the third domain comprising the amino acid sequence selectedfrom any one of SEQ ID NOs: 181-188.
 19. The method of claim 18, whereinsaid antibody construct comprises a polypeptide comprising the aminoacid sequence of SEQ ID NO:
 160. 20. A method for treating glioblastomaor glioma, comprising administering to a subject in need thereof aneffective amount of a polypeptide comprising: (a) a first binding domainthat binds to human and macaque Epidermal Growth Factor receptor VIII(EGFRVIII), and comprises: a heavy chain variable region (VH) comprisingthe amino acid sequence of SEQ ID NO: 157, and a light chain variableregion (VL) comprising the amino acid sequence of SEQ ID NO: 158; and(b) a second binding domain that binds to human CD3, and comprises: a VLregion comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 92,a CDR-L2 comprising the amino acid of SEQ ID NO:93, and a CDR-L3comprising the amino acid of SEQ ID NO:94; and a VH region comprising: aCDR-H1 comprising the amino acid of SEQ ID NO: 95, a CDR-H2 comprisingthe amino acid of SEQ ID NO:96, and a CDR-H3 comprising the amino acidof SEQ ID NO:97.
 21. The method of claim 20, wherein said polypeptidecomprises (a) a first binding domain that binds to human and macaqueEGFRVIII, and comprises: a VH comprising the amino acid sequence of SEQID NO: 157, and a VL comprising the amino acid sequence of SEQ ID NO:158; and (b) a second binding domain which binds to human CD3, andcomprises a VH comprising the amino acid sequence of SEQ ID NO: 98, anda VL comprising the amino acid sequence of SEQ ID NO:
 99. 22. The methodof claim 20, wherein said polypeptide comprises (a) a first domaincomprising the amino acid sequence of SEQ ID NO: 159; and (b) a seconddomain comprising the amino acid sequence of SEQ ID NO:
 100. 23. Themethod of claim 20, wherein said polypeptide further comprises a His-tagcomprising the amino acid sequence of SEQ ID NO:
 10. 24. The method ofclaim 20, wherein said polypeptide comprises the amino acid sequence asset forth in SEQ ID NO:
 189. 25. The method of claim 20, wherein saidpolypeptide comprises the amino acid sequence as set forth in SEQ ID NO:190.
 26. A method for treating glioblastoma or glioma, comprisingadministering to a subject in need thereof an effective amount of apolypeptide comprising an amino acid sequence of SEQ ID NO:
 160. 27. Themethod of claim 26, wherein said polypeptide further comprises a His-tagcomprising the amino acid sequence of SEQ ID NO:
 10. 28. The method ofclaim 26, wherein said polypeptide comprises in the following orderstarting from the N-terminus: an amino acid sequence as set forth in SEQID NO: 160, and a His-tag as set forth in SEQ ID NO:
 10. 29. The methodof claim 26, wherein said polypeptide further comprises a single-chainFc (scFc) comprising an amino acid sequence selected from any one of SEQNOs: 181-188.
 30. The method of claim 29, wherein said polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO:
 189. 31.The method of claim 29, wherein said polypeptide comprises the aminoacid sequence as set forth in SEQ ID NO:
 190. 32. The method of claim20, wherein said polypeptide is administered intravenously (IV).
 33. Themethod of claim 26, wherein said polypeptide is administeredintravenously (IV).
 34. The method of claim 26, wherein said polypeptideis administered by continuous intravenous (IV) administration.